JP2023015311A - Multimer, tetramer and octamer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multimer of an effector domain.

SOLUTION: The present invention relates to a multimer such as a tetramer and an effector domain (antigen-binding site (antibody or TCR binding site specifically binding to antigen or pMHC, or its variable domain) or the like, for example) of polypeptide, or a tetramer and an octamer of peptide such as incretin, insulin or hormone peptide.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention provides tetramers of polypeptides as well as effector domains (such as antigen binding sites (e.g., antibodies or TCR binding sites that specifically bind antigen or pMHC, or variable domains thereof)) or incretin, insulin or hormone peptides. Tetramers and multimers such as octamers of peptides such as

Multimers of effector domains have recognized utility in medical and non-medical applications for combining and doubling the activity and presence of effector domains, such as (e.g., antibodies or TCR binding domains biological activity or avidity, such as to provide higher avidity of antigen binding (for effector domains that are ) or to provide bi- or multi-specific targeting or interaction with target ligands in vivo or in vitro It is intended to promote

Multimerization domains that cause the self-assembly of protein monomers into multimers are known in the art. Examples include domains found in transcription factors such as p53, p63 and p73, as well as domains found in ion channels such as the TRP cation channel. Transcription factor p53 can be divided into different functional domains: N-terminal transactivation domain, proline-rich domain, DNA binding domain, tetramerization domain and C-terminal regulatory region. The tetramerization domain of human p53 extends from residues 325-356 and has a 4-helical bundle fold (Jeffrey et al., Science (New York, N.Y.) 1995, 267(5203):1498- 1502). The TRPM tetramerization domain is a short antiparallel coiled-coil tetramerization domain of transient receptor potential cation channel subfamily M member proteins 1-8. It is held together by extensive core packing and interstrand polar interactions (Fujiwara et al., Journal of Molecular Biology 2008, 383(4):854-870). Transient receptor potential (TRP) channels constitute a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Another example is the BTB domain of potassium channels. This domain can be found at the N-terminus of voltage-gated potassium channel proteins and represents the cytoplasmic tetramerization domain (T1) responsible for assembly of the alpha subunit into functional tetrameric channels (Bixby et al. ., Nature Structural Biology 1999, 6(1):38-43). This domain is also found in proteins that are not potassium channels, such as KCTD1 (potassium channel tetramerization domain-containing protein 1; Ding et al., DNA and Cell Biology 2008, 27(5):257-265). be able to.

Multimeric antibody fragments have been produced using a variety of multimerization techniques, such as biotin, dHLX, ZIP and BAD domains, as well as p53 (Thie et al., Nature Boitech., 2009:26, 314- 321). Efficient to manufacture, biotin is a bacterial protein that induces an immune response in humans.

Human p53 (UniProtKB-P04637 (P53_HUMAN)) acts as a tumor suppressor in many tumor types, inducing growth arrest or apoptosis depending on the physiological milieu and cell type. It participates in cell cycle regulation as a transactivator and acts to negatively regulate cell division by regulating a set of genes required for this process. Human p53 is found in increased amounts in a variety of transformed cells. It is frequently mutated or inactivated in approximately 60% of cancers. Deficiency of human p53 is seen in Barrett's metaplasia, a condition in which the normally stratified squamous epithelium of the lower esophagus is replaced by metaplastic columnar epithelium. This condition develops as a complication with chronic gastroesophageal reflux disease in approximately 10% of patients and predisposes to the development of esophageal adenocarcinoma.

Nine isoforms of p53 exist naturally and are expressed in a wide range of normal tissues in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues, but is not detected in brain, lung, prostate, muscle, fetal brain, spinal cord and fetal liver. Isoform 3 is expressed in most normal tissues, but is not detected in lung, spleen, testis, fetal brain, spinal cord and fetal liver. Isoform 7 is expressed in most normal tissues but not detected in prostate, uterus, skeletal muscle and breast. Isoform 8 is detected only in colon, bone marrow, testis, fetal brain and intestine. Isoform 9 is expressed in most normal tissues, but is not detected in brain, heart, lung, fetal liver, salivary gland, breast or intestine.

The present invention provides:

In a first configuration, a protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., protein domain or peptide), wherein the multimers associate together in the first , second, third and fourth self-associating tetramerization domains (TD), each tetramerization domain carrying one or more copies of said protein domain or peptide A multimer composed of each engineered polypeptide comprising:

In a second configuration: an isolated tetramer or octamer, or a plurality of said tetramers or octamers, of a TCR binding site, insulin peptide, incretin peptide or peptide hormone.
An isolated tetramer or octamer, or a plurality of said tetramers or octamers, of an antibody binding site or antibody variable domain (eg, a single variable domain).
In one example, the tetramer or octamer is soluble in aqueous solutions (eg, aqueous eukaryotic cell culture media). In one example, tetramers or octamers can be expressed in eukaryotic cells. Examples are provided below.

In a third configuration: (a) a TCR V domain or TCR binding site tetramer or octamer that is soluble in an aqueous solution (e.g., an aqueous eukaryotic cell growth medium or buffer);
(b) tetramers or octamers of antibody single variable domains that are soluble in aqueous solutions (e.g. aqueous eukaryotic cell growth media or buffers);
(c) a tetramer or octamer of a TCR V domain or TCR binding site that can be expressed intracellularly and/or extracellularly by HEK293 cells, or (d) intracellularly and/or extracellularly by HEK293 cells Tetramers or octamers of antibody variable domains (eg antibody single variable domains) that are expressible.

In a fourth configuration, a multimeric, tetrameric or octameric engineered polypeptide or monomer of the invention.

In the fifth configuration (in the N-terminal to C-terminal direction)
(a) TCR V1-TCR C1-antibody CH1 (eg IgG CH1)-optionally present linker-including TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Cβ;
(iii) V1 is Vγ and C1 is Cγ, or (iv) V1 is Vδ and C1 is Cδ,
or (b) TCR V1-antibody CH1 (eg IgG CH1)-optionally present linker-TD,
(i) V1 is Vα;
(ii) V1 is Vβ;
(iii) V1 is Vγ, or (iv) V1 is Vδ,
or (c) antibody V1-antibody CH1 (e.g. IgG CH1)-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (d) antibody V1 - optionally present antibody CH1 (e.g. IgG CH1) - antibody Fc (e.g. IgG Fc) - optionally present linker - TD,
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (e) comprising antibody V1-antibody CL (e.g., Cλ or Cκ)-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (f) TCR V1-TCR C1-optionally present linker-TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Cβ;
(iii) V1 is Vγ and C1 is Cγ, or (iv) V1 is Vδ and C1 is Cδ,
Engineered (and optionally isolated) engineered polypeptide (P1).

In a sixth configuration, a nucleic acid encoding an engineered polypeptide or monomer of the invention, optionally constituted by an expression vector for expressing said polypeptide.

In a seventh configuration, use of the nucleic acid or vector of the present invention in a method for producing a protein multimer for producing a multimer that is expressed and/or secreted into a cell, wherein the method uses the nucleic acid or vector expressing said multimer in a eukaryotic cell comprising and/or secreting said multimer from said eukaryotic cell.

In an eighth configuration (a) comprises soluble and/or intracellular expression of a TCR V-TD (e.g., NHR2 TD or TCR V-p53 TD) fusion protein expressed in a eukaryotic cell, optionally comprising multiple a method of producing a TCR V domain multimer comprising isolating said multimer;
(b) soluble and/or intracellular expression of an antibody V (e.g., single variable domain)-TD (e.g., V-NHR2 TD or V-p53 TD) fusion protein expressed in eukaryotic cells, where A method of producing antibody V domain multimers, comprising isolating a plurality of said multimers by
(c) soluble and/or intracellular expression of an incretin peptide-TD (e.g., incretin peptide-NHR2 TD or incretin peptide-p53 TD) fusion protein expressed in eukaryotic cells such as HEK293T cells; (d) in eukaryotic cells such as HEK293T cells soluble and/or intracellular expression of the expressed peptide hormone-TD (e.g., peptide hormone-NHR2 TD or peptide hormone-p53 TD) fusion protein, and optionally isolating a plurality of said multimers. , Methods for Producing Peptide Hormone Multimers.

In a ninth configuration, use of a nucleic acid or vector of the invention in a method for producing protein multimers for producing glycosylated multimers in a eukaryotic cell comprising said nucleic acid or vector.

In a tenth configuration, a self-associating tetramerization domain ( TD) (eg, NHR2 TD, p53 TD, p63 TD or p73 TD or homologs or orthologs thereof).

In an eleventh configuration, in a method for producing a tetramer of a polypeptide comprising multiple copies of a protein domain or peptide, for producing the tetramer with a higher yield than monomeric and/or dimeric polypeptides. Use of an engineered polypeptide, wherein the engineered polypeptide comprises one or more copies of said protein domain or peptide and a self-associating tetramerization domain (TD) (e.g. NHR2 TD , p53 TD, p63 TD or p73 TD or homologs or orthologs).

In a twelfth configuration self-associating tetramerization in a method for producing tetramers of a polypeptide to produce a plurality of tetramers that are not mixed with monomers, dimers or trimers Use of domains (TDs) such as NHR2 TD, p53 TD, p63 TD or p73 TD or homologs or orthologs thereof.

In a thirteenth configuration, a eukaryotic host cell comprising a nucleic acid or vector for intracellular and/or secretory expression of a multimer, tetramer, octamer, engineered polypeptide or monomer of the invention.

In the fourteenth configuration

Engineered methods for producing tetramers of a polypeptide comprising multiple copies of a protein domain or peptide to produce tetramers that are not mixed with monomers, dimers or trimers. and wherein the engineered polypeptide comprises one or more copies of said protein domain or peptide and contains a self-associating tetramerization domain (TD) (e.g. NHR2 TD, p53 TD, p63 TD or p73 TD or homologs or orthologs).

In a fifteenth configuration, (i) a TCR extracellular domain,
A multivalent heterodimeric soluble T-cell receptor capable of binding pMHC complexes, comprising (ii) an immunoglobulin constant domain, and (iii) the NHR2 multimerization domain of ETO.

In a sixteenth configuration, a multimeric immunoglobulin comprising (i) an immunoglobulin variable domain and (ii) an NHR2 multimerization domain of ETO.

In a seventeenth configuration, a method of assembling into soluble multimeric polypeptides, comprising:
(a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO;
(b) a method comprising associating multiple copies of said monomers, thereby obtaining a multimeric soluble polypeptide.

In an eighteenth configuration, (i) a cell line encoding a polypeptide of the invention (e.g., a eukaryotic mammalian cell line, e.g., HEK293, CHO or Cos cell line), and (ii) a tetramer of the invention A mixture, including the body.

In a nineteenth configuration, a method of enhancing the yield of a tetramer of a protein effector domain (e.g. antibody variable domain or binding site), said method comprising converting the tetramer of the polypeptide into a cell line (e.g. animal cells, CHO, HEK293 or Cos cell lines), said polypeptide is a polypeptide of the invention and comprises one or more effector domains, and said method optionally comprises , isolating said expressed tetramer.

The invention also provides pharmaceutical compositions, cosmetics, foodstuffs, beverages, cleaning products, cleaning agents comprising the multimers, tetramers or octamers of the invention.

Schematic representation of the stepwise self-assembly of a tetravalent heterodimeric soluble TCR protein complex via monomers and homodimers assisted by the NHR2 tetramerization domain. A construct representing the stepwise self-assembly of the octavalent heterodimeric soluble TCR protein complex via monomer ² and homodimer ² , assisted by the NHR2 tetramerization domain and the immunoglobulin hinge domain. It is a diagram. FIG. 2 is a schematic diagram of the domain arrangement of the a and β chains used to express and assemble the ts-NY-ESO-1 TCR. FIG. 2 is a schematic diagram of the domain arrangement of the a and β chains used to express and assemble the os-NY-ESO-1 TCR. Amino acid sequences of the a and β chains of the ts-NY-ESO-1 TCR protein complex. The amino acid sequences of alternating domains are underlined. Amino acid sequences of the a and β chains of the os-NY-ESO-1 TCR protein complex. The amino acid sequences of alternating domains are underlined. Schematic representation of the domain arrangement of the a and β chains used to express and assemble the ts-NY-ESO-1 TCR-IL2 fusion protein complex. FIG. 2 is a schematic diagram of the a and β chain domain arrangements used to express and assemble the os-NY-ESO-1 TCR-IL2 fusion protein complex. Amino acid sequences of the a and β chains of the ts-NY-ESO-1 TCR-IL2 fusion protein complex. The amino acid sequences of alternating domains are underlined. Amino acid sequences of the a and β chains of the os-NY-ESO-1 TCR-IL2 fusion protein complex. The amino acid sequences of alternating domains are underlined. Schematic representation of the stepwise self-assembly of tetravalent single domain antibody (dAb) complexes via monomers and homodimers assisted by the NHR2 tetramerization domain. Schematic diagram of the domain arrangement for assembly of tetravalent dAbs, including linker and NHR2 domains. Schematic representation of the stepwise self-assembly of tetravalent Fab complexes via monomers and homodimers assisted by the NHR2 tetramerization domain. Schematic diagram of the domain arrangement for assembly of tetravalent Fabs, including the linker and NHR2 domains in the heavy chain and the light chain variable and constant domains. Schematic representation of the stepwise self-assembly of octavalent Fab complexes via monomers and homodimers assisted by antibody hinge regions linked to NHR2 tetramerization domains and CH1 domains. . Schematic diagram of the domain arrangement for the assembly of octavalent Fabs, including the hinge, linker and NHR2 domains in the heavy chain and the light chain variable and constant domains. FIG. 2 is a configuration diagram of Quad 16 and Quad 17; (A) Quad 16 and (B) Quad 17 monomer sequences and configurations are shown. Ditto Ditto Ditto Analysis of secreted proteins using anti-Ig western blot is shown: (A) PAGE gel under SDS denaturing conditions-16=Quad16; 17=Quad17; and (B) PAGE under native (i.e. non-denaturing) conditions. Gel-16=Quad16; 17=Quad17. Western blot prepared from a denaturing SDS-PAGE gel probed with an anti-human IgG HRP detection antibody. (A) Protein samples from Quad 3 and Quad 4 were prepared from whole cell extracts and run in lanes 1 and 2, respectively. The predicted Mw for Quad 3 and Quad 4 are 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quad 12 and Quad 13 were prepared from whole cell extracts and run in lanes 1 and 2, respectively. The predicted Mw for Quad 12 and Quad 13 are 47.8 and 48.1 kDa, respectively. Western blot prepared from a denaturing SDS-PAGE gel probed with an anti-human IgG HRP detection antibody. (A) Protein samples from Quad 3 and Quad 4 were prepared by concentrating cell supernatants and run in lanes 1 and 2, respectively. The predicted Mw for Quad 3 and Quad 4 are 46.1 and 46.4 kDa, respectively. (B) Protein samples from Quad 12 and Quad 13 were prepared by concentrating cell supernatants and run in lanes 1 and 2, respectively. The predicted Mw for Quad 12 and Quad 13 are 47.8 and 48.1 kDa, respectively. Western blot prepared from a denaturing SDS-PAGE gel probed with an anti-HIS HRP detection antibody. (A) Protein samples from Quad 14, Quad 15, Quad 18 and Quad 19 were prepared from whole cell extracts and run in lanes 1-4, respectively. The predicted Mw for Quad 14, Quad 15, Quad 18 and Quad 19 are 22.0, 22.3, 37.4 and 37.7 kDa, respectively. (B) Protein samples from Quad 23, Quad 24, Quad 26 and Quad 27 were prepared from whole cell extracts and run in lanes 1-4, respectively. The predicted Mw for Quad 23, Quad 24, Quad 26 and Quad 27 are 32.1, 32.4, 33.7 and 34.0 kDa, respectively. (C) Protein samples from Quad 34, and Quad 38 were prepared from whole cell extracts and run in lanes 1-2, respectively. The predicted Mw for Quad 34 and Quad 38 are 25.5 and 25.4 kDa, respectively. (D) Protein samples from Quad 40, Quad 42, Quad 44 and Quad 46 were prepared from whole cell extracts and run in lanes 1-4, respectively. The predicted Mw for Quad 40, Quad 42, Quad 44 and Quad 46 are 25.4, 37.6, 25.5 and 38.0 kDa, respectively. Lane U contains concentrated serum prepared from untransfected HEK293T cells (negative control), C is His-tagged protein used as positive control for anti-His HRP detection antibody. A serum anti-His background band is highlighted by a black arrow and can be detected consistently in all blots. Ditto (A) Western blot prepared from a denaturing SDS-PAGE gel and probed with an anti-human IgG HRP detection antibody. Protein samples from Quad 14 and Quad 15 were prepared from whole cell extracts and run in lanes 1 and 2, respectively. The predicted Mw for Quad 14 and Quad 15 are 22.0 and 22.3 kDa, respectively. (B) Western blot prepared from a native PAGE gel probed with an anti-human IgG HRP detection antibody. Lanes 1 and 2 contain protein samples from Quad 14 and Quad 15 prepared from whole cell extracts. Quad polyeptides fused to leader and tag sequences. If a linker is present, the linker is a G4S (one G4S only). * indicates TCR constant domains that have been introduced with cysteine residues that allow the formation of S—S bonds between the alpha and beta chains of the TCR. A human IgG1 hinge was used. All C regions are human. The TCR V domain is specific for NY-ESO-1. GFP = green fluorescent protein. Ditto Id. All polypeptide diagrams and amino acid sequences herein are written from the N-terminus to the C-terminus. All nucleotide sequences herein are written from 5' to 3'.

DETAILED DESCRIPTION The present invention provides polypeptide tetramers and effector domains (such as antigen binding sites (e.g., antibodies or TCR binding sites that specifically bind antigen or pMHC, or variable domains thereof)) or incretins, insulin or multimers such as tetramers and octamers of peptides such as hormone peptides. In embodiments, the multimers of the present invention are useful for manufacturing in eukaryotic systems and are useful in various industrial applications, such as manufacturing pharmaceuticals, diagnostics, imaging agents, cleaning agents, etc. The form can be secreted from eukaryotic cells. Higher order multimers such as tetramers or octamers of effector domains or peptides are useful for enhancing antigen or pMHC binding avidity. This may be useful for the production of effective agents or for enhancing the sensitivity of diagnostic reagents containing multimers, tetramers or octamers. An additional or alternative advantage is that the in vivo half-life is enhanced when the multimers of the invention are administered to a human or animal subject, e.g., to treat or prevent a disease or condition in the subject. is. Usefully, the present invention can also provide multispecific (eg, bi- or tri-specific) multivalent binding proteins. Specificity can relate to specificity of antigen or pMHC binding. By using a single engineered polypeptide comprising a binding domain or peptide, the present invention, in certain instances, usefully produces highly pure and multivalent (e.g., bispecific) proteins. Provide means for manufacturing. The use of a single species of engineered polypeptide monomer is useful when two or more different polypeptide species are used to produce a multi (e.g., bi)specific or multivalent protein. Avoids the mixed product problem seen.

The present invention provides the following clauses, aspects and concepts. Any clause in this specification can be combined with any aspect or concept in this specification. Any aspect in this specification can be combined with any concept in this specification.

Aspect:
1. A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., protein domain) or peptide, wherein said multimers associate together each engineered protein multimerizing by a third and fourth self-associating tetramerization domain (TD), each tetramerization domain comprising one or more copies of said protein domain or peptide; A multimer composed of multiple polypeptides.

In one example, each TD is a TD of any one of proteins 1-119 listed in Table 2. In one example, each TD is a p53 TD or homologue or ortholog thereof. In one example, each TD is an NHR2 TD or homologue or ortholog thereof. In one example, each TD is a p63 TD or homologue or ortholog thereof. In one example, each TD is a p73 TD or homologue or ortholog thereof. In one example, each TD is not an NHR2 TD. In one example, each TD is not a p53 TD. In one example, each TD is not a p63 TD. In one example, each TD is not a p73 TD. In one example, each TD is not a p53, 63 or 73 TD. In one example, each TD is not an NHR2, p53, 63 or 73 TD.

"Associating together" means that the TD of embodiment 1 multimerizes the first, second, third and fourth copies of the engineered polypeptide to form a multimeric protein, e.g. can be expressed intracellularly in cells or mammalian cells (e.g. HEK293 cells) and/or can be secreted extracellularly from eukaryotic or mammalian cells (e.g. HEK293 cells) and/or in aqueous media (e.g. To provide multimers that are soluble in eukaryotic or mammalian cell (eg, HEK293 cell) culture medium). Examples thereof are NHR TD, p53 TD, p63 TD and p73 TD (eg human NHR TD, p53 TD, p63 TD and p73 TD) or orthologs or homologs thereof.

In one example, the TD is not p53 TD (or its homologues or orthologs), eg, it is not human p53 TD (or its homologues or orthologs). In one example, the TD is an NHR2 TD or homologue or ortholog thereof, but not a p53 TD or a homologue or ortholog thereof. In one example, the TD is a human NHR2 TD or its homologues or orthologs, but not a human p53 TD or its homologues or orthologs. In one example, the TD is human NHR2. In one example, the amino acid sequence of TD is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of human NHR2. In one example, the domain or peptide is not naturally constituted by a polypeptide that also contains an NHR2 TD.

In one example, all of the domains of the polypeptide are human.

The engineered polypeptide may comprise one or more copies of said domain or peptide N-terminal to said copy of TD. Additionally or alternatively, the engineered polypeptide may comprise one or more copies of said domain or peptide at the C-terminus of said copy of TD. In one example, the engineered polypeptide comprises a first first said domain or peptide and a TD, wherein the first domain or peptide is at least 10, 20, 30, 40, 50, 60, 70, from the TD. 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous amino acids apart and between the first domain or peptide and the TD a further said domain or peptide does not exist.

In one example, a multimer (e.g., a tetramer of said engineered polypeptide) comprises 4 (but no more than 4) TDs (e.g., identical TDs) and 4, 8, 12 or 16 ( provided that it contains no more than 4, 8, 12 or 16 copies of said domain or peptide, respectively. In one example, each TD and each said domain or peptide is human.

In one example, a multimer, tetramer or octamer comprises first, second, third and fourth identical copies of an engineered polypeptide, wherein the polypeptide is a TD and one (provided , but not more than one), two (but not more than two), or more copies of said protein domain or peptide.

In some embodiments, by requiring only one engineered polypeptide to form a multimer, tetramer or octamer of the invention, the invention advantageously provides: In pure (or highly pure, i.e. greater than 90, 95, 96, 97, 98 or 99% pure) formats, such A method of manufacturing a format is provided. Purity is defined as not mixed in composition with any other multimers or polypeptide monomers or other multimers comprising the multimers of the invention and engineered polyeptides. and/or comprising more than 90, 95, 96, 97, 98 or 99% of the species in the composition comprising the polypeptide monomers. Thus, mixtures of different types of polypeptides are avoided or minimized in these embodiments. Thus, it is also advantageously a plurality of multimers (e.g. a plurality of tetramers or octamers) comprising only one (not more than one) type of engineered polypeptide, Multimers are provided that are monospecific (but multivalent) for antigen binding, or alternatively bi- or multispecific for antigen binding. Thus, the present invention provides multiple multimers (e.g., multiple tetramers or octamers), each polypeptide being at least tetravalent for antigen binding, and (i) bispecific (i.e., capable of specifically binding to two different antigens), or (ii) is monospecific and at least tetravalent for antigen binding.When antigen binding is described herein, Where the domain is a TCR V domain this may alternatively be pMHC binding Advantageously the plurality is in pure form, i. (e.g., tetramers or octamers).In one example, the multimers comprise at least two different types of antigen binding sites.In one example, the multimers are bispecific In one example, the multimer has a valency of 4, 6, 8, 10 or 12, preferably 4 or 8 antigen or pMHC binding sites.

In one example, the peptide MHC (pMHC) is class I or class II pMHC.

For example, the term "specifically binds" as used herein with respect to a domain, antibody or binding site means that the domain, antibody or binding site binds with a binding affinity of 1 mM or less as determined by SPR. It means recognizing a specific antigen (or pMHC).

Target binding capacity, specificity and affinity (KD (also referred to as Kd), K _off and/or K _on ) are determined by any routine method in the art, e.g. surface plasmon resonance (SPR) be able to. As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant of a particular binding site/ligand, receptor/ligand or antibody/antigen interaction. In one embodiment, surface plasmon resonance (SPR) is performed at 25°C. In another embodiment, SPR is performed at 37°C. In one embodiment, SPR is performed at physiological pH, eg, about pH 7 or pH 7.6 (eg, using Hepes-buffered saline at pH 7.6 (also referred to as HBS-EP)). In one embodiment, SPR is performed at physiological salt levels, eg, 150 mM NaCl. In one embodiment, the SPR is 0.05% by volume or less in the presence of detergent levels, such as 0.05% P20 (polysorbate 20; eg, Tween-20™) and 3 mM EDTA. executed. In one example, SPR is performed at 25° C. or 37° C. in a pH 7.6 buffer, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Buffers may contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25°C or 37°C. HBS-EP is available from Teknova Inc (California; catalog number H8022). In one example, affinities (eg, affinities of VH/VL binding sites) are measured using any standard SPR instrument such as the Biacore™ or ProteOn XPR36™ (Bio-Rad® )) is determined using SPR by using Binding data can be fitted to a unique 1:1 model using standard techniques, eg, using the model unique to ProteOn XPR36™ analysis software.

In one example, a multimer, tetramer or octamer of the invention is an isolated multimer, tetramer or octamer. In one example, a multimer, tetramer or octamer of the invention consists of copies of said engineered polypeptide. Optionally, the multimers, tetramers or octamers of the invention comprise 4 or 8 but no more than 4 or 8 copies of the engineered polypeptide, respectively.

"Engineered" means that the polypeptide does not occur in nature, eg, the protein domain or peptide is not naturally constituted by a polypeptide that also includes said TD.

Each said protein domain or peptide may be a biologically active domain or peptide (e.g. biologically active in humans or animals), e.g. domain, or a domain constituted by an antigen or pMHC binding site. Alternatively, the domain or peptide is a carbohydrate, glucose or sugar regulator, such as an incretin or insulin peptide. Alternatively, the domains or peptides are inhibitors or enzymes or inhibitors of biological functions or pathways in humans or animals. Alternatively, the domain or peptide is an iron modulating agent. Thus, in one example, each protein domain or peptide is selected from antigens or pMHC binding domains or peptides; hormones; carbohydrate, glucose or sugar regulators; iron regulators; and enzyme inhibitors.
2. A multimer of any preceding aspect, which is a tetramer or octamer of said domains or peptides.
3. 3. The multimer of any aspect 1 or 2, comprising a tetramer or octamer of immunoglobulin superfamily binding sites (eg, antibody or TCR binding sites, such as scFv or scTCR).

The immunoglobulin superfamily (IgSF) is a large protein superfamily of cell-surface and soluble proteins involved in cell recognition, binding, or adhesion processes. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins (also known as antibodies), and they all possess domains known as immunoglobulin domains or folds. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular muscle proteins. mentioned. They are usually associated with roles in the immune system.

The T cell receptor (TCR) domain may be Vα (e.g., pairing with Vβ), Vβ (e.g., pairing with Vα), Vy (e.g., pairing with Vδ) or Vδ (e.g., pairing with Vy). possible.
4. 4. The multimer of aspect 3, wherein said binding site comprises a first variable domain paired with a second variable domain.

In a first example, the first and second variable domains are composed of engineered polypeptides. In another example, the first domain is composed of the engineered polypeptide and the second domain is different from the engineered polypeptide (and optionally comprises or comprises a TD). missing) by additional polypeptides.

Alternatively, the domains are constant region domains. Alternatively, the domain is an FcAb. Alternatively, the domain is a non-Ig antigen binding site or is constituted by a non-Ig antigen binding site, such as an affibody.

Antigen-binding site and effector domains In one example, the or each antigen-binding site (or effector domain) is an antibody variable domain (e.g., VL or VH, antibody single variable domain (domain antibody or dAb), camelid VHH antibody single single variable domain, shark immunoglobulin single variable domain (NAV), Nanobody™ or camelized VH single variable domain); T-cell receptor binding domain; immunoglobulin superfamily domain; 2010 Aug l;185(3):1367-74; "Alternative adaptive immunity in jawless vertebrates; Herrin BR & Cooper M D.); fibronectin domains (e.g., Adnectin™); scFv; scFv)2; sc-diabody; scFab; scaffold-derived antigen-binding site (Evibody™) selected from sentinin and CTLA-4; lipocalin domain; ™ or SpA); A domains (e.g. Avimer™ or Maxibody™); heat shock proteins (such as epitope binding domains from GroEI and GroES); transferrin domains (e.g. transbodies); ankyrin repeats proteins (e.g., DARPins™); peptide aptamers; C-type lectin domains (e.g., Tetranectin™); human γ-crystallin or human ubiquitin (affilins); PDZ domains; is selected from the group consisting of type domains;

Further sources of antigen binding sites are the antibody variable domains and VH/VL pairs disclosed on page 40, line 23 to page 43, line 23 of WO2007024715. This particular disclosure as expressly set forth herein to provide a basis for epitope binding moieties for use in the present invention and to allow for inclusion in the claims of this application. , which is incorporated herein by reference.

A "domain" is a folded protein structure that has tertiary structure independent of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or domains. can. A "single antibody variable domain" is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. Thus, single antibody variable domains include complete antibody variable domains and modified variable domains, e.g., those in which one or more loops are replaced by sequences not characteristic of antibody variable domains, or truncated Antibody variable domains that are modified or contain N- or C-terminal extensions, as well as folded fragments of the variable domains that retain at least the binding activity and specificity of the full-length domain.

The phrases "immunoglobulin single variable domain" or "antibody single variable domain" refer to antibody variable domains (VH, VHH, VL) that specifically bind antigens or epitopes independent of different V regions or domains. An immunoglobulin single variable domain can exist in a format (e.g. homo- or heteromultimers) having other different variable regions or variable domains, said other regions or domains being associated with a single immunoglobulin variable domain. Not required for antigen binding (ie, the immunoglobulin single variable domain binds antigen independently of additional variable domains). A "domain antibody" or "dAb", as the term is used herein, is the same as an "immunoglobulin single variable domain" capable of binding an antigen. The immunoglobulin single variable domain can be a human antibody variable domain, but single antibody variable domains from other species such as rodents (such as those disclosed in WO 00/29004). ), nurse shark and camelid VHH immunoglobulin single variable domains. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species such as camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains can be humanized according to standard techniques available in the art and such domains are still considered "domain antibodies" according to the present invention. As used herein, "VH" includes camelid VHH domains. NAVs are another type of immunoglobulin single variable domain identified in cartilaginous fish such as nurse sharks. is also known as the Novel Antigen Receptor variable region (commonly abbreviated as V (NAR) or NARV) For further details see Mol. Immunol. ) and US Patent Application Publication No. 20050043519. CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. The domains have a variable domain-like Ig fold Loops corresponding to antibody CDRs can be replaced with heterologous sequences to confer different binding properties CTLA-4 engineered to have different binding specificities The molecule is also known as Evibody For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001) Lipocalins are hydrophobic small molecules such as steroids, bilins, retinoids and lipids. A family of extracellular proteins that transport molecules They possess a rigid β-sheet secondary structure with a number of loops at the free end of a conical structure that can be manipulated to bind different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins, for further details see Biochim Biophys Acta 1482: 337-350 (2000), US Pat. Affibodies are scaffolds derived from protein A of Staphylococcus aureus that can be engineered to bind antigens.The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues For further details see Protein Eng. Des. Sel. reference. Avimers™ are multidomain proteins derived from the A-domain scaffold family. The approximately 35 amino acid natural domain adopts a defined disulfide bond structure. Diversity is generated by shuffling the natural variations exhibited by families of A domains. For further details see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999). Designed Ankyrin Repeat Proteins (DARPins™) are derived from the ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two a-helices and a β-turn. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and β-turn of each repeat. Their binding interfaces can be increased by increasing the number of modules (method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007), and US See Patent Application Publication No. 20040132028. Fibronectin is a scaffold that can be engineered to bind antigen. Adnectins™ consist of the natural amino acid sequence backbone of the tenth domain of the fifteen repeat units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to allow Adnectins to specifically recognize therapeutic targets of interest. For further details see Protein Eng. Des. Sel. 18, 435-444 (2005), US Patent Application 20080139791, WO2005056764 and US Pat. No. 6,818,418. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), which contains a constrained variable peptide loop inserted into the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from small naturally occurring proteins of 25-50 amino acids in length containing 3-4 cysteine bridges; examples of small proteins include KalataBI and conotoxins and knottins. Small proteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall fold of the small protein. See WO2008098796 for further details of engineered knotting domains. Other epitope binding moieties and domains include proteins that have been used as scaffolds to engineer different target antigen binding properties, human γ-crystallin and human ubiquitin (affilin), Kunitz forms of human protease inhibitors PDZ domain of Ras-binding protein AF-6, scorpion toxin (chalybdotoxin), C-type lectin domain (tetranectin), which are described in Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel ), Chapter 7 and Protein Science 15:14-27 (2006).
5. Each polypeptide comprises first and second copies of said protein domain or peptide, said polypeptide comprising (in N-terminal to C-terminal direction): (i) said first copy - TD - said second (ii) TD- and said first and second copies; or (iii) said first and second copies-TD.
6. Any preceding wherein said TD is an NHR2 TD and said domain or peptide is not an NHR2 domain or peptide; or said TD is a p53 TD and said domain or peptide is not a p53 domain or peptide Embodiment multimers.
7. said engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, wherein said second type of protein domain or peptide is different from said first protein domain or peptide , the multimer of any preceding aspect.

For example, the polypeptide comprises, in the N-terminal direction, (i) P1-TD-P2; or (ii) TD-P1-P2, wherein P1 is the domain of the first type (i.e. P2 is a copy of a domain or peptide of the second type) and P2 is a copy of said second type of domain or peptide.
8. The multimer of any preceding aspect, wherein said domain is an immunoglobulin superfamily domain.
9. The multimer of any preceding aspect, wherein said domains or peptides are antibody variable or constant domains (eg antibody single variable domains), TCR variable or constant domains, incretins, insulin peptides, or hormone peptides.
10. said multimer comprises first, second, third and fourth identical copies of said engineered polypeptide, said polypeptide comprising a TD and one of said protein domains or peptides, provided that A multimer of any preceding aspect, including no more than 1), 2 (but no more than 2) or more copies.
11. The multimer of any preceding aspect, wherein said engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD.
12. (ii) V1-optional linker-NHR2 TD-optional linker-V2 or (iii) V1-optional linker-V2-optional linker-NHR2 TD, wherein V1 and V2 are TCR variable domains and are the same or different, or V1 and V2 are antibody 12. A multimer according to embodiment 11, which is a variable domain and is the same or different.
13. 13. The multimer of embodiment 12, wherein V1 and V2 are antibody single variable domains.
14. each engineered polypeptide comprises (from N-terminus to C-terminus) V1-optionally present linker-NHR2 TD, where V1 is an antibody or TCR variable domain, and each engineered polypeptide is paired with each second engineered polypeptide comprising V2, wherein V2 is an antibody or TCR variable domain that pairs with V1, respectively, to form an antigen or pMHC binding site; and Optionally, the multimer of aspect 11, wherein one polypeptide comprises the antibody Fc or comprises the antibody CH1 and the other polypeptide comprises the antibody CL paired with CH1.
15. said TD comprises (i) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 10 or 126; or (ii) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 120 or 123 , the multimer of any preceding aspect.
16. Rituxan™; Rituximab; Zenapax™; Daclizumab; Simulect™; Remicade™; Infliximab; Herceptin™; Mylotarg™; Gemtuzumab; Campath™; Humira(TM); Adalimumab; Xolair(TM); Omalizumab; Bexxar(TM); bevacizumab; Tysabri™; natalizumab; Actemra™; tocilizumab; Vectibix™; eculizumab; Cimzia™; certolizumab; Simponi™; golimumab, Ilaris™; Ofatumumab; Prolia™; Denosumab; Numax™; Motavizumab; ABThrax™; ipilimumab; Adcetris™; brentuximab; Vedotin™; Perjeta™; ad trastuzumab; Keytruda™, Opdivo™, Gazyva™ and obinutuzumab antigen binding of an antibody selected from the group consisting of A multimer of any preceding aspect, including a tetramer or octamer of the sites.

For example, said protein domain of the engineered polypeptide is the V domain (VH or VL) of an antibody binding site of an antibody selected from said group and the multimer pairs with the V domain of the engineered polypeptide. and an additional V domain (VL or VH, respectively) that forms the antigen-binding site of the selected antibody. Advantageously, therefore, the present invention provides a tetramer or octamer of the binding site of said selected antibody, which advantageously binds to its cognate antigen, Alternatively, multimers may be administered to have improved affinity, avidity and/or efficacy for treating or preventing diseases or conditions in humans or animals that bind cognate antigens in vivo.

For example, a multimer, tetramer or octamer comprises four copies of the antigen-binding site of an antibody, wherein the antibody is adalimumab, sarilumab, dupilumab, bevacizumab (e.g. Avastin™), cetuximab (e.g. Erbitux ™), tocilizumab (eg Actemra™) or trastuzumab (Herceptin™). Alternatively, the antibody is an anti-CD38 antibody, an anti-TNFa antibody, an anti-TNFR antibody, an anti-IL-4Ra antibody, an anti-IL-6R antibody, an anti-IL-6 antibody, an anti-VEGF antibody, an anti-EGFR antibody, an anti-PD-1 antibody. , anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PCSK9 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD138 antibody, anti-IL-1 antibody. Alternatively, the antibody is selected from those disclosed on page 40, line 23 to page 43, line 23 of WO2007024715, the disclosure of which is incorporated herein by reference. be done.

A binding site in the present invention may be, for example, a ligand (eg, cytokine or growth factor, eg, VEGF or EGFR) binding site of a receptor (eg, KDR or Flt). For example, the binding site in the present invention may be the binding site of Eyelea™, Avastin™ or Lucentis™, for example for eye or tumor medical use in humans or animals. . When the ligand or antigen is VEGF, mutlimers, tetramers or octamers are produced in cancer or eye conditions (e.g., wet or dry AMD or diabetic retina in human or animal subjects). disease) or as an inhibitor of angiogenesis.
17. An isolated tetramer or octamer, or a plurality of said tetramers or octamers, of a TCR binding site, insulin peptide, incretin peptide or peptide hormone.

A number of important peptide hormones are secreted by the pituitary gland. The anterior pituitary gland secretes three hormones: prolactin, which acts on the mammary gland; adrenocorticotropic hormone (ACTH), which acts on the adrenal cortex and regulates glucocorticoid secretion; and growth hormone, which acts on bone, muscle, and liver. . The posterior pituitary gland secretes the antidiuretic hormone, also called vasopressin, and oxytocin. Peptide hormones are produced by many different organs and tissues, including the heart (atrial natriuretic peptide (ANP) or atrial natriuretic factor (ANF)) and pancreas (glucagon, insulin and somatostatin), Includes gastrointestinal tract (cholecystokinin, gastrin), and adipose tissue storage (leptin). In one example, the peptide hormones of the invention are selected from prolactin, ACTH, growth hormone (somatotropin), vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin (e.g. human prolactin, ACTH, growth hormone, vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin).

In one example, the incretin is a GLP-1, GIP or exendin-4 peptide.

The invention provides, in embodiments, the following engineered tetramers and octamers:
Isolated tetramers or octamers of incretins.
An isolated tetramer or octamer of an insulin peptide.
GLP-1, an isolated tetramer or octamer of the glucagon-like peptide-1 (GLP-1) peptide.
An isolated tetramer or octamer of GIP (glucose-dependent insulinotropic polypeptide) peptide.
An isolated tetramer or octamer of an exendin (eg, exendin-4) peptide.
Isolated tetramers or octamers of peptide hormones.
An isolated tetramer or octamer of prolactin or prolactin peptides.
An isolated tetramer or octamer of ACTH or an ACTH peptide.
An isolated tetramer or octamer of growth hormone or a growth hormone peptide.
An isolated tetramer or octamer of vasopressin or a vasopressin peptide.
An isolated tetramer or octamer of oxytocin or an oxytocin peptide.
An isolated tetramer or octamer of glucagon or a glucagon peptide.
An isolated tetramer or octamer of insulin or insulin peptide.
An isolated tetramer or octamer of somatostatin or a somatostatin peptide.
An isolated tetramer or octamer of cholecystokinin or a cholecystokinin peptide.
An isolated tetramer or octamer of gastrin or a gastrin peptide.
An isolated tetramer or octamer of leptin or a leptin peptide.
An isolated tetramer or octamer of an antibody binding site (eg, scFv or Fab).
An isolated tetramer or octamer of a TCR binding site (eg, scTCR).
Isolated tetramers or octamers of the TCR Vα/Vβ binding sites.
Isolated tetramers or octamers of the TCR Vy/Vδ binding site.
Isolated tetramers or octamers of antibody single variable domain binding sites.
Isolated tetramers or octamers of FcAb binding sites.

In one example of any of these tetramers or octamers, the domains or peptides are human. In one example of any of these tetramers or octamers, the tetramer or octamer includes an NHR2 TD (eg, human NHR2). In one example of any of these tetramers or octamers, the tetramer or octamer includes p53 TD (eg, human p53 TD). In one example of any of these tetramers or octamers, the tetramer or octamer comprises a p63 TD (eg, human p63 TD). In one example of any of these tetramers or octamers, the tetramer or octamer comprises a p73 TD (eg, human p73 TD). In one example of any of these tetramers or octamers, the tetramer or octamer comprises a tetramer of TD (e.g., human NHR2 TD), whereby the domain or peptide consists of four or form multimers of eight domains or peptides.

In one example, the plurality is pure and not mixed with multimers of said binding sites or peptides, eg comprising more than one type of polypeptide monomer.
18. (a) is soluble in aqueous solutions (e.g., aqueous eukaryotic cell growth media or buffers);
(b) secretable from a eukaryotic cell and/or (c) an expression product of a eukaryotic cell,
A multimer, tetramer or octamer of any preceding aspect.

In one example, multimers, tetramers or octamers are detected using a sample of supernatant from HEK293T (or other eukaryotic, mammalian, CHO or Cos) cells and using a Western blot. It can be secreted from such cells in a stable form indicated by a single band of molecular weight predicted for said multimer, tetramer or octamer on a PAGE gel.
19. (a) tetramers or octamers of TCR V domains or TCR binding sites that are soluble in aqueous solutions (e.g., aqueous eukaryotic cell growth media or buffers);
(b) tetramers or octamers of antibody single variable domains that are soluble in aqueous solutions (e.g. aqueous eukaryotic cell growth media or buffers);
(c) a tetramer or octamer of a TCR V domain or TCR binding site that can be expressed intracellularly and/or extracellularly by HEK293 cells, or (d) intracellularly and/or extracellularly by HEK293 cells Tetramers or octamers of antibody variable domains (eg antibody single variable domains) that are expressible.

An example medium is SFMII growth medium supplemented with L-glutamine (eg, complete SFMII growth medium supplemented with 4 mM L-glutamine). In one example, the medium is serum-free HEK293 cell culture medium. In one example, the medium is serum-free CHO cell culture medium.

For example, cells in the present invention are human cells, such as HEK293 cells (such as HEK293T cells).
20. The multimer, tetramer or octamer of any preceding aspect, wherein said tetramer or octamer is bispecific for antigen or pMHC binding.
21. The multimer, tetramer or octamer of any preceding aspect, wherein said domains are identical.
22. A multimer, tetramer or octamer of any preceding aspect, including eukaryotic glycosylation.

For example, the glycosylation is that of CHO cells. For example, the glycosylation is HEK (eg, HEK293, such as HEK293T) cell glycosylation. For example, the glycosylation is Cos cell glycosylation. For example, the glycosylation is that of Picchia cells. For example, the glycosylation is that of Saccharomyces cells.
23. 23. The multimer, tetramer or octamer of embodiment 22, wherein said cells are HEK293 cells.
24. A plurality of multimers, tetramers or octamers of any preceding aspect.
25. A pharmaceutical composition comprising a multimer, tetramer or octamer of any preceding aspect and a pharmaceutically acceptable carrier, diluent or excipient.
26. Cosmetics, foodstuffs, beverages, cleaning products, cleaning agents comprising the multimer, tetramer or octamer of any one of aspects 1-24.
27. Said engineered (and optionally isolated) polypeptide or (optionally isolated) monomer of a multimer, tetramer or octamer of any preceding aspect.

A monomer is an engineered polypeptide disclosed herein comprising said protein domain or peptide and further comprising a TD.

Optionally, the engineered polypeptide comprises (from N-terminus to C-terminus) a variable domain (V1) - a constant domain (C) (eg CH1 or Fc) - an optional linker - TD.
28. (N-terminal to C-terminal direction):
(a) TCR V1-TCR C1-Antibody C (e.g., CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ))-optionally present linker-TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Vβ;
(iii) V1 is Vy and C1 is Cy, or (iv) V1 is Vδ and C1 is C8,
or (b) TCR V1-antibody C (e.g., CH, CH1 (such as IgG CH1) or CL (such as Cλ or Cκ))-optionally present linker-TD,
(i) V1 is Vα;
(ii) V1 is Vβ;
(iii) V1 is Vy, or (iv) V1 is Vδ,
or (c) antibody V1-antibody C (e.g., CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ))-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (d) antibody V1 - optionally present antibody C (e.g. CH, CH1 (such as IgG CH1) or CL (such as Ck or Cκ)) - antibody Fc (e.g. IgG Fc) - optionally present linker - TD including
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (e) comprising antibody V1-antibody CL (e.g. Ck or Cκ)-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL (e.g., Vλ or Vκ),
or (f) TCR V1-TCR C1-optionally present linker-TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Cβ;
(iii) V1 is Vγ and C1 is Cγ, or (iv) V1 is Vδ and C1 is Cδ,
Engineered (and optionally isolated) engineered polypeptide (P1).

In (a) or (b), in one example, TCR V is constituted by a single-chain TCR binding site (scTCR) that specifically binds to pMHC, said binding site comprising TCR V-linker-TCRV. In one example, the engineered polypeptide comprises (from N-terminus to C-terminus) (i) V1-linker-V-optionally C-optionally linker-TD, or (ii) Vα-linker -V1-optional C-optional linker-TD with Vα being a TCR V domain and C being an antibody C domain (eg CH1 or CL) or TCR C.

Preferably, antibody C is CH1 (eg IgG CH1).

In one example the multimer, tetramer or octamer has a size of 155 kDa or less, e.g. An antibody variable domain comprising a CDR3 of 20, 21 or 22 amino acids.

In one example, a multimer, tetramer or octamer includes a TCR binding site and an antibody binding site. For example, each polypeptide may comprise a TCR V (e.g., composed of a scTCR that specifically binds pMHC) and an antibody V (e.g., scFv), or a second polypeptide composed of said second polypeptide. V domain to form a V/V paired binding site that specifically binds antigen). In one example, the pMHC contains a RAS peptide. In one example, the antigen is selected from the group consisting of PD-1, PD-L1, or any other antigen disclosed herein. For example, the antigen is PD-1 and the pMHC contains the RAS peptide.
29. Said engineered polypeptide P1 is paired with a further polypeptide (P2), P2 (in the N-terminal to C-terminal direction):
(g) TCR V2-TCR C2-Antibody CL (e.g., Cλ or Cκ), wherein P1 is according to (a) as described in aspect 28, and (i) P1 is (a)(ii) V2 is Vα and C2 is Cα if by
(ii) if P1 is according to (a)(i), then V2 is Vβ and C2 is Cβ;
(iii) if P1 is due to (a)(iv), then V2 is Vγ and C2 is Cγ, or (iv) if P1 is due to (a)(iii), V2 is Vδ and C2 is Cδ,
or (h) TCR V2-antibody CL (e.g., Ck or Cκ), wherein P1 is according to (b) as described in aspect 28, and (i) P1 is according to (b)(ii) , then V2 is Vα, and
(ii) V2 is Vβ if P1 is according to (b)(i);
(iii) V2 is Vγ if P1 is due to (b)(iv), or (iv) V2 is Vδ if P1 is due to (b)(iii);
or (i) comprising antibody V2-CL (e.g., Ck or Cκ), P1 is according to (c) as described in aspect 28, and (i) P1 is according to (c)(ii) In some cases V2 is VH, or (ii) V2 is VL (e.g., Vλ or VK) if P1 is according to (c)(i);
or (j) antibody V2 - optionally comprising a CL (e.g., Ck or Cκ), wherein P1 is according to (d) as described in aspect 28, and (i) P1 is (d)(ii) ) if V2 is VH, or (ii) V2 is VL (e.g., Vλ or Vκ) if P1 is due to (d)(i);
or (k) comprising antibody V2-CH1 (e.g., IgG CH1), wherein P1 is according to (e) as described in aspect 28, and (i) P1 is according to (e)(ii) if V2 is VH, or (ii) V2 is VL (e.g., Vλ or Vκ) if P1 is due to (e)(i);
or (l) comprises TCR V2-TCR C2, wherein P1 is according to (f) as described in aspect 28, and (i) P1 is according to (f)(ii), where V2 is Vα and C2 is Cα,
(ii) V2 is Vβ and C2 is Cβ if P1 is according to (f)(i);
(iii) if P1 is due to (f)(iii), then V2 is Vγ and C2 is Cγ, or (iv) if P1 is due to (f)(iv), V2 is Vδ and C2 is Cδ,
29. The polypeptide of embodiment 28.

Optionally, V1 and V2 form a paired variable domain binding site capable of specifically binding antigen or pMHC. In one example, V1 and V2 are, for example, Leopro™; Abciximab; Rituxan™; Rituximab; Xenapax™; Gemtuzumab; Campus™; Alemtuzumab; Zevalin™; Ibritumomab; Humira™; Adalimumab; Cetuximab; Avastin™; Bevacizumab; Tysabri™; Natalizumab; Actemra™; Tocilizumab; Cimzia™; Certolizumab; Simponi™; Golimumab, Ilaris™; Canakinumab; Belimumab; Yervoy™; Ipilimumab; ADCETRIS™; Brentuximab; Ad-trastuzumab; Keytruda™, Opdivo™, Gazyva™ and obinutuzumab.

In one embodiment, the antibody is Avastin.

In one embodiment, the antibody is Actemra.

In one embodiment, the antibody is Erbitux.

In one embodiment, the antibody is Lucentis.

In one embodiment, the antibody is sarilumab.

In one embodiment, the antibody is dupilumab.

In one embodiment, the antibody is alirocumab.

In one embodiment, the antibody is evolocumab.

In one embodiment, the antibody is pembrolizumab.

In one embodiment, the antibody is nivolumab.

In one embodiment, the antibody is ipilimumab.

In one embodiment, the antibody is Remicade.

In one embodiment, the antibody is golimumab.

In one embodiment, the antibody is ofatumumab.

In one embodiment, the antibody is Benlysta.

In one embodiment, the antibody is campus.

In one embodiment, the antibody is rituximab.

In one embodiment, the antibody is Herceptin.

In one embodiment, the antibody is durvalumab.

In one embodiment, the antibody is daratumumab.

In one example V1 is (by itself if a single variable domain or when paired with V2) ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; AIG1; AKAP1; AKAP2; AIYIH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; BAI1; BCL2; BCL6; BDNF; BLNK; (IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1;CCBP2 (D6/JAB61); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (M IP-3b); CCL2 (MCP-1); CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 I Eotaxin-2); CCL25 (TECK); CCL26 (Eotaxin-3); CCL28; CCL3 (MIP-la); CCL4 (MIP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; EBI1); CCR8 (CM KBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD20; CD200; CD-22; CD24; CD28; CD3; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; CSF3 (GCSF); CTLA4; CTNNBl (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDi); CX3CR1 (V28); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; EN01; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FLTl; FOS; FOSLl (FRA-I); FY (DARC); GABRP (GABAa); GAGEB1; GPR31; GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (gelsolin); IFNA1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFN gamma;TFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2 IL10RA; IL10RB; IL11; IL11RA; IL-12; IL17R;1L18;IL18BP;IL18R1;IL18RAP;1L19;ILIA;IL1B;IL1F10;IL1F5;IL1F6;IL1F7; IL20RA;IL21R;1L22;1L22R;1L22RA2;1L23;1L24;1L25 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; INSL3; INSL4; IRAKI; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6 integrin); KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); MIP-2; MK167 (Ki-67); MMP2; M MP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF1 1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFSFIO (TRAIL); TNFSF1 1 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-lBB ligand); TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM 1; TREM2; TRPC6; TSLP;
/CCXCR1); YY1; and ZFPM2.

For example, in any configuration of the invention, the multimer, tetramer or octamer specifically binds to first and second epitopes or antigens, each of said first and second epitopes or antigens VEGF and VEGFR2; VEGF and EGFR; CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and CD138; CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTN02; CSPG and RGM A; IGF1 and IGF2; IL-13 and ADAM8; IL-13 and CL25; IL-13 and IL-lbeta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; RGM A and RGM B; TNF alpha and IL-12p40; TNF alpha and IL-13; TNF alpha and IL-15; TNF alpha and IL-17; TNF alpha and IL-18; TNF alpha and MIF; TNF alpha and PEG2; TNF alpha and PGE4; TNF alpha and VEGF; and VEGFR and EGFR; CD-22; and selected from the group consisting of TNF alpha and CTLA-4.

For example, the first epitope or antigen is selected from the group consisting of CD3; CD16; CD32; CD64; and CD89, and the second epitope or antigen is EGFR; CD33; CD79a; CD19; PSA; EpCAM; .

In one example, V1 (by itself as a single variable domain or when paired with V2) is human IL-1A, IL-1β, IL-1RN, IL-6, BLys, APRIL, capable of specifically binding to an antigen selected from the group consisting of activin A, TNF alpha, BMP, BMP2, BMP7, BMP9, BMP10, GDF8, GDF11, RANKL, TRAIL, VEGFA, VEGFB or PGF, optionally The multimer comprises an amino acid sequence (e.g., C-terminal to TD) of a cytokine, such as IL-2 or IL2-peptide, and the multimer, tetramer or octamer treats cancer in a human subject. or preventive. In one example, said effector or protein domain is capable of binding such an antigen, and optionally the multimer comprises an amino acid sequence (e.g., C-terminal to TD) of a cytokine, such as IL-2 or IL2-peptide. and the multimer, tetramer or octamer is for treating or preventing cancer in a human subject.
30. P1 as defined in aspect 28, or multimers of P1 paired with P2 as defined in aspect 29 (e.g. dimers, trimers, tetramers or octamers), or a plurality of said multimers and optionally said multimer is according to any one of aspects 1-24.

Preferably, the multimer is a tetramer of engineered polypeptides and/or effector domains. In one example, tetramers are not mixed with monomers, dimers or trimers of the polypeptide.

In one example, a multimer, eg, a tetramer, can specifically bind to two different pMHCs.
31. A nucleic acid encoding the engineered polypeptide or monomer of any one of aspects 27-29, optionally wherein said nucleic acid is constituted by an expression vector for expressing said polypeptide .

In one example, the nucleic acid is DNA, optionally operably linked to or including a promoter for expression of the polypeptide or monomer. In another example, the nucleic acid is RNA (eg, mRNA).
32. a nucleic acid or vector of aspect 31 for intracellular and/or secretory expression of a multimer, tetramer, octamer, engineered polypeptide or monomer of any one of aspects 1-24. nuclear host cell.
33. 32. Use of the nucleic acid or vector according to aspect 31 in a method for producing protein multimers for producing intracellularly expressed and/or secreted multimers, said method comprising a eukaryotic protein comprising said nucleic acid or vector A use comprising expressing said multimer in a cell and/or secreting said multimer from said eukaryotic cell.
34. Use of a nucleic acid or vector according to aspect 31 in a method for producing protein multimers for producing glycosylated multimers in a eukaryotic cell comprising said nucleic acid or vector.

Glycosylation of a mammal of the invention produces a medicament comprising or consisting of a multimer, tetramer or octamer of the invention for the medical treatment or prevention of a disease or condition in a mammal, e.g. a human. useful for Accordingly, the invention provides multimers, tetramers or octamers of the invention for this purpose, as well as methods of such use. Similarly, intracellular and/or secretory expression in one or more mammalian host cells (or cell lines thereof) according to the present invention is useful for producing such agents. Such expression in HEK293, CHO or Cos cells is particularly useful as these cells are commonly used for the production of pharmaceuticals.

In one embodiment, the invention includes cleaning agents or personal health care products comprising the multimers, tetramers or octamers of the invention. In one embodiment, the invention includes foodstuffs or beverages comprising the multimers, tetramers or octamers of the invention.

In one example, the multimers, monomers, dimers, trimers, tetramers, octamers, polypeptides, compositions, mixtures, uses or methods of the invention are suitable for industrial or domestic use. or used in a method for such use. For example, it is used in agriculture, oil or petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, Medical industry, healthcare industry, dental industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry , the clothing industry, the leather or suede or hide industry, the tobacco industry or the steel industry.
35. (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of aspects 27-29; and (ii) a multimer, tetramer as defined in any one of aspects 1-24. A mixture containing isomers or octamers.
36. 36. The mixture of aspect 35, wherein said cell line is in a medium comprising secreted products of said cells, said secreted products comprising said multimers, tetramers or octamers.
37. A multimer, tetramer or octamer of any one of aspects 1-24 for medical use.
38. (a) soluble and/or intracellular expression of a TCR V-NHR2 TD or TCR V-p53 TD fusion protein expressed in a eukaryotic cell, optionally comprising isolating a plurality of said multimers; , a method for producing TCR V domain multimers;
(b) comprising soluble and/or intracellular expression of antibody V (e.g. single variable domain)-NHR2 TD or V-p53 TD fusion proteins expressed in eukaryotic cells, optionally a plurality of said multimers; A method of producing antibody V domain multimers comprising isolating
(c) soluble and/or intracellular expression of an incretin peptide-NHR2 TD or incretin peptide-p53 TD fusion protein expressed in eukaryotic cells such as HEK293T cells, optionally comprising a plurality of said multimers; (d) a peptide hormone-NHR2 TD expressed in eukaryotic cells such as HEK293T cells, or A method of producing peptide hormone multimers comprising soluble and/or intracellular expression of a peptide hormone-p53 TD fusion protein and optionally isolating a plurality of said multimers.
39. A self-associating tetramerization domain (TD) (e.g., NHR2 TD, p53 TD, p63 TD or p73 TD or homologues or orthologs thereof).
40. Engineered Polypeptides in Methods for Producing Tetramers of Polypeptides Comprising Multiple Copies of a Protein Domain or Peptide to Produce Tetramers in Higher Yields than Monomeric and/or Dimeric Polypeptides wherein said engineered polypeptide comprises one or more copies of said protein domain or peptide and comprises a self-associating tetramerization domain (TD) (e.g. NHR2 TD, p53 TD, p63 TD or p73 TD or homologs or orthologs).
41. A self-associating tetramerization domain (TD) ( For example, use of NHR2 TD, p53 TD, p63 TD or p73 TD or homologues or orthologues thereof).
42. Engineered methods for producing tetramers of a polypeptide comprising multiple copies of a protein domain or peptide to produce tetramers that are not mixed with monomers, dimers or trimers. wherein said engineered polypeptide comprises one or more copies of said protein domain or peptide and contains a self-associating tetramerization domain (TD) (e.g. NHR2 TD, p53 TD, p63 TD or p73 TD or homologs or orthologs).
43. 43. Use according to any one of aspects 39-42, wherein said yield of tetramer is at least 10, 20, 30, 40 or 50 times said yield of monomer and/or dimer.
44. the ratio of tetramer produced in the method to monomer and/or dimer produced is at least 90:10 (e.g., at least 95:5 or 98:2, or 99:1); Use of any one of aspects 39-43.
45. 45. Use according to any one of aspects 39-44, wherein each monomer has a size of 40, 35, 30, 25 or 20 kDa or less.
46. 46. Use according to any one of aspects 39-45, wherein each tetramer has a size of 200, 160, 155 or 150 kDa or less.
47. 47. The use of any one of aspects 39-46, wherein said method comprises expressing said tetramer from a eukaryotic cell line.
48. (i) a TCR extracellular domain;
A multivalent heterodimeric soluble T-cell receptor capable of binding pMHC complexes, comprising (ii) an immunoglobulin constant domain, and (iii) the NHR2 multimerization domain of ETO.
49. A multimeric immunoglobulin comprising (i) an immunoglobulin variable domain and (ii) an NHR2 multimerization domain of ETO.
50. A method of assembling into soluble multimeric polypeptides, comprising:
(a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO;
(b) a method comprising associating multiple copies of said monomers, thereby obtaining a multimeric soluble polypeptide.

The present invention further provides:
(i) a monomer shown in FIG. 1;
(ii) the homodimer shown in Figure 1;
(iii) the homotetramer shown in FIG. 1;
(iv) monomer ^{2 shown in Figure 2} ;
(v) homodimer 2 shown in Figure ² ;
(vi) homotetramer 2 shown in Figure ² ;
(vii) the monomers shown in Figure 11a;
(viii) the homodimer shown in Figure 11a;
(ix) the homotetramer shown in Figure 11a;
(x) a monomer shown in Figure 12a;
(xi) the homodimer shown in Figure 12a;
(xii) the homotetramer shown in Figure 12a;
(xiii) monomer ² shown in Figure 13a;
(xiv) homodimer ² shown in Figure 13a;
(xv) homotetramer ² shown in FIG. 13a; or (xvi) any one of (i)-(xv) (e.g., Quad 3, Quad 4, Quad 12, Quad 13, Quad 14, Quad 15 , Quad 16 and Quad 17), or multimers of any of the proteins shown in FIG. 21 (excluding any leader or tag).
(xvii) a plurality of multimers of (xvi); or (xviii) a pharmaceutical composition comprising any one of (i)-(xvii) and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides:
(i) a tetravalent or octavalent antibody V molecule;
(ii) tetravalent or octavalent antibody Fab molecules;
(iii) a tetravalent or octavalent antibody dAb molecule;
(iv) tetravalent or octavalent antibody scFv molecules;
(v) a tetravalent or octavalent antibody TCR V molecule; or (vi) a tetravalent or octavalent antibody scFv molecule, said molecule comprising
(a) soluble in aqueous solutions (eg, solutions or cell culture media disclosed herein); and/or (b) intracellularly and/or extracellularly expressible by HEK293 cells.

The present invention claims NHR2 or p53 (or another TD disclosed herein) fused at its N-terminus and/or C-terminus to an amino acid sequence (e.g., a peptide, protein domain or protein) that is not an NHR2 sequence. Multimers (eg, tetramers) of terms are provided. For example, sequences may be TCRs (eg TCRa, TCRβ, Ca or Oβ), cytokines (eg interleukins such as IL-2, IL-12, IL-12 and IFN), antibody fragments (eg scFv, dAb or Fab) and antibody domains (eg V or C domains such as VH, VL, Vκ, Vλ, CH, CH1, CH2, CH3, hige, Cκ or Cx domains). Optionally, the multimer is
(i) soluble in aqueous solutions (e.g., solutions or cell culture media disclosed herein) and/or (ii) intracellularly and/or extracellularly expressible by HEK293 cells,
is a molecule.

The present invention inventonprovides:
(i) NHR2 or p53 (or p53 herein) for the production of polypeptides for soluble expression of multimers of the polypeptides from cells, such as eukaryotic cells, such as mammalian, HEK293, CHO or Cos cells; Use of another TD) disclosed in .
(ii) NHR2 or p53 (or the present Use of another TD) disclosed in the specification.
(iii) a cell containing an intracellular expression product, NHR2 fused at its N-terminus and/or C-terminus to an amino acid sequence (e.g., a peptide, protein domain or protein) that is not an NHR2 sequence; or a cell comprising a multimer of a polypeptide comprising p53 (or another TD disclosed herein).
(iv) as a promiscuous tetramerization domain for tetramerizing peptides, protein domains, polypeptides or proteins in the production of multimers that are intracellularly and/or solublely expressed from host cells; use of NHR2.

Optionally, the amino acids are human peptides, protein domains or proteins such as TCRs (eg TCRa, TCRβ, Ca or Cβ), cytokines (eg interleukins such as IL-2, IL-12, IL-12 and IFN), antibody fragments (e.g. scFv, dAb or Fab), or antibody domains (e.g. V or C domains such as VH, VL, Vκ, Vλ, CH, CH1, CH2, CH3, hinge, Cκ or Cλ domain) amino acid sequence.

Optionally, the or each polypeptide comprises a polypeptide selected from the group consisting of Quads 1-46 (ie, the polypeptides shown in Figure 21, but excluding any leader or tag sequences). Optionally, the invention consists of such Quads 1-46, eg for medical or diagnostic use, eg for treating or preventing a disease or condition in humans or animals (eg humans). Polypeptide multimers selected from the group (e.g. dimers, trimers, tetramers, pentamers, hexamers, heptamers or octamers, preferably tetramers or octamers) )I will provide a.

Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequences) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOS: 13-50. Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequences) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:83-115. Optionally, the invention provides multimers of such polypeptides ( For example, dimers, trimers, tetramers, pentamers, hexamers, heptamers or octamers, preferably tetramers or octamers).

In one example, the TD is a TD made up of any one of SEQ ID NOs: 1-9. In one example, the TD is a TD comprising SEQ ID NO: 10 or 126. In one example, TD is encoded by SEQ ID NO: 124 or 125. In one example, the amino acid sequence of each TD is SEQ ID NO: 10 or 126, or is at least 80, 85, 90, 95, 96m 97, 98 or 99% identical to SEQ ID NO: 10 or 126.

In one example, the TD is a TD comprising SEQ ID NO: 120 or 123. In one example, TD is encoded by SEQ ID NO: 116 or 119. In one example, the amino acid sequence of each TD is SEQ ID NO: 120 or 123, or is at least 80, 85, 90, 95, 96m 97, 98 or 99% identical to SEQ ID NO: 120 or 123.

Optionally, the domain or peptide constituted by the engineered polypeptide or monomer comprises amino acids selected from SEQ ID NOs:51-82.

High Purity Tetramers As exemplified herein, the present invention, in one configuration, uses a tetramerization domain (TD), such as a p53 tetramerization domain (p53 TD), to form a dimer Based on the surprising realization that tetramers of effector domains can be produced preferentially over the production of lower order structures such as isomers and monomers. This is particularly useful because tetramer secretion is a desirable yield from mammalian expression cell lines such as CHO, HEK293 and Cos cell lines. The invention is also particularly useful for the production of tetramers of sizes 200, 160, 155 or 150 kDa or less.

Accordingly, the present invention provides the following concepts:
concept
1. A tetramerization domain (TD) (e.g., p53 tetramer use of the transcoding domain (p53 TD) or NHR2 TD) or homologues or orthologues thereof.

Monomers and dimers contain 1 or 2 copies of TD, homologs or orthologs, respectively.

In one example, the TDs, orthologs or homologues are human domains.

In one example, the tetramer yield is higher than the monomer yield; in one example, the tetramer yield is higher than the dimer yield; , higher than trimer yields; in one example, tetramer yields are higher than monomer and dimer yields; in one example, tetramer yields are higher than monomer and trimer yields; Higher than the yield of the mer; in one example, the yield of the tetramer is higher than the yield of the monomer, dimer and trimer.

For example, the TD is the TD of p53 isoform 1. In one example, a TD comprises or consists of an amino acid sequence identical to positions 325-356 (or 319-360; or 321-359) of human p53 (eg, isoform 1). Optionally, a TD, ortholog or homologue comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10, 126, 11 or 12. For example, the sequences are identical to the selected sequences. Optionally, a TD, ortholog or homologue comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 120, 121, 122 or 123. For example, the sequence is identical to the selected sequence.
2. Use of concept 1, wherein the first, second, third and fourth copies of the same TD or its homologues or orthologs are used.
3. Use of any of the preceding concepts, wherein said TD is the tetramerization domain of NHR2, p53, p63 or p73.
For example, TD is p53 TD. In one example, the TD is an ortholog or homologue of p53 TD, eg, human p53 TD.
4. Use of any preceding concept wherein said yield of tetramer is at least 10 times said yield of monomer and/or dimer.

Optionally, the yield is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the yield of monomer and/or dimer . Optionally, the tetramer:monomer and/or dimer ratio produced is at least 90:10, such as at least 95:5; or 96:4; or 97:3; or 98:2; or 99:1. In some cases only tetramers are produced.

In one embodiment, each domain constituted by each monomer, dimer or tetramer is a human domain; and optionally the monomer, dimer or tetramer is a non-human amino acid sequence or linker.
5. The ratio of tetramer produced in the method to monomer and/or dimer produced is at least 90:10 (i.e., 9 times the amount of monomer; 9 times the amount of dimer or 9 times the amount of the combination of monomers and dimers).
6. obtaining a sample of the product of the tetramer manufacturing process in which said yield or ratio is obtained; separating tetramers, monomers and dimers using protein separation techniques on said sample; and the use of concept 4 or 5, determinable or determined by comparing the amount of tetramer with the amount of monomer and dimer.

The amounts of tetramers, monomers, dimers and trimers are not under denaturing conditions, such as in the gels described herein, e.g., native gels, i.e., in the absence of SDS. Western blot analysis of gels can be used to determine.
7. wherein said yield or ratio is
(a) obtaining a sample of the product of the tetramer manufacturing process;
(b) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to reveal a band corresponding to said tetramer and a band corresponding to said monomer and/or a band corresponding to said dimer; separating said sample; and (c) comparing said tetramer band to said monomer and/or dimer band, e.g., by comparing relative band intensities and/or band sizes. Use of concept 4 or 5, determinable or determined by determining said yield or ratio.
8. wherein said yield or ratio is
(d) obtaining a sample of the product of the tetramer manufacturing process;
(e) performing polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions to separate said sample into bands corresponding to said tetramers, e.g., said gel under non-denaturing conditions (e.g. in the absence of sodium dodecylsulfate (SDS)), separating
(f) Use of concept 4 or 5, which is determinable or determined by determining the absence of a band corresponding to said monomer and/or a band corresponding to said dimer.
9. (g) obtaining a second sample of the product of the tetramer manufacturing process;
(h) performing polyacrylamide gel electrophoresis (PAGE) under reducing conditions to separate said second sample into bands corresponding to said monomers and/or bands corresponding to said dimers; separating, e.g., said gel is under non-denaturing conditions (e.g., in the absence of sodium dodecyl sulfate (SDS)); and (i) the gel produced by step (h). determining the position of the monomer and/or dimer bands in the gel of step (b) compared to the gel of b) or (e); Use of concept 7 or 8, including determining what is expected to be the gel of e).
10. Use of any preceding concept in which each monomer has a size of 40 kDa or less.

For example, the monomer has a size of 35, 30, 25, 24, 23, 22, 21 or 20 kDa or less.
11. Use of any preceding concept wherein each tetramer has a size of 150 kDa or less.

For example, a tetramer has a size of 80, 90, 100, 110, 120, 130 or 140 kDa or less.
12. Use of any preceding concept wherein said method comprises expressing said tetramer from a mammalian cell line, such as HEK293, CHO or Cos cell lines.

For example, the cell line is the HEK293 (eg HEK293T) cell line. Alternatively, the cell line is a yeast (eg Saccharomyces or Pichia, eg P pastoris) or bacterial cell line.
13. Use of any preceding concept wherein said method comprises secreting said tetramer from a mammalian cell line, such as HEK293, CHO or Cos cell lines.

Advantageously, therefore, in one example the use or tetramer is for expression from a mammalian cell line (eg HEK293, CHO or Cos cell lines) or a eukaryotic cell line. This is useful for large-scale production of products of the invention, such as tetramers.

For example, the cell line is the HEK293 (eg HEK293T) cell line. Alternatively, the cell line is a yeast (eg Saccharomyces or Pichia, eg P pastoris) or bacterial cell line.
14. Each polypeptide or monomer forms an antigen binding site with said TD, homolog or ortholog, and one or more protein effector domains, such as one or more antibody domains, e.g., one or more antibodies use of any preceding concept, including domains).
15. said polypeptide is
(i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding an antigen;
(ii) scFv capable of binding antigen or scTCR capable of binding pMHC;
(iii) a Fab capable of binding antigen, or (iv) a use of concept 14 comprising one or more of a TCR variable domain or pMHC binding site.
16. Use of any of the preceding concepts wherein each polypeptide or monomer comprises said TD, homolog or ortholog, and one or more incretin, insulin, GLP-1 or exendin-4 domains.
17. Use of any of the preceding concepts wherein each polypeptide or monomer comprises said TD, homolog or ortholog, and first and second antigen binding sites.
18. Each binding site is
(i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding an antigen;
(ii) scFv capable of binding antigen or scTCR capable of binding pMHC;
Use of concept 17 provided by (iii) a Fab capable of binding antigen, or (iv) a TCR variable domain or pMHC binding site.
19. Use of concept 18, wherein each binding site is provided by an antibody single variable domain.
20. Use of any one of concepts 14-18, wherein said TD, homolog or ortholog is fused directly to said additional domain.
21. Use of concept 20, wherein each monomer or polypeptide comprises said TD, homolog or ortholog fused to the C-terminus of said further domain either directly or via a peptide linker.
22. A tetramer of polypeptides, each polypeptide comprising:
(i) a tetramerization domain (TD) (e.g. p53 TD or NHR2 TD) or homologues or orthologs thereof;
(ii) one or more protein effector domains, and (iii) optionally a linker linking (i) to (ii) (e.g. linking the C-terminus of (ii) to the N-terminus of (i)) including
optionally each tetramer has a size of 150 or 200 kDa or less,
Tetramer.

For example, a tetramer has a size of 80, 90, 100, 110, 120, 130 or 140 kDa or less. In one example, any multimer, dimer, trimer, tetramer or octamer in the present invention has a size of at least 60 or 80 kDa, which is associated with, for example, disease in a subject or to increase half-life in a human or animal subject administered a multimer, dimer, trimer, tetramer or octamer (to treat or prevent a condition). The magnitude of these ranges can be greater than that of renal filtration.

Alternatively, the invention provides monomers, dimers, tetramers or octamers instead of tetramers.
23. each polypeptide
(i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding an antigen;
(ii) scFv capable of binding antigen or scTCR capable of binding pMHC;
(iii) a Fab capable of binding antigen, or (iv) a tetramer of concept 22 comprising one or more of a TCR variable domain or pMHC binding site.
24. The tetramer of concept 22 or 23, wherein each polypeptide comprises said TD, homolog or ortholog, and one or more incretin, insulin, GLP-1 or exendin-4 domains.
25. The tetramer of concept 22 or 23, wherein each polypeptide comprises said TD, homolog or ortholog, and first and second antigen binding sites.
26. Each binding site is
(i) an antibody single variable domain (dAb or VHH or Nanobody™) capable of specifically binding an antigen;
(ii) scFv capable of binding antigen or scTCR capable of binding pMHC;
(iii) a Fab capable of binding antigen, or (iv) a tetramer of concept 25 provided by a TCR variable domain or pMHC binding site.
27. A tetramer of Concept 26, wherein each binding site is provided by an antibody single variable domain.
28. A tetramer of any one of concepts 22-27, wherein said TD, homolog or ortholog is fused directly to said effector domain.
29. The tetramer of any one of concepts 22-27, wherein each polypeptide comprises said TD, homolog or ortholog fused to the C-terminus of said effector domain either directly or via a peptide linker.

In one embodiment, each polypeptide has only two effector domains (i.e. only the first and second and not the third) or only two dAbs, VHHs, scFv, scTCR, Fab or antigen Contains the binding site.
30. A pharmaceutical composition comprising a tetramer of any one of Concepts 22-29 and a pharmaceutically acceptable carrier, diluent or excipient.

Optionally, the composition is contained in a sterile medical container or device such as a syringe, vial, inhaler or injection device.
31. Cosmetics, foodstuffs, beverages, cleaning products, cleaning agents comprising a tetramer of any one of concepts 22-29.
32. cell lines (e.g., mammalian cell lines such as HEK293, CHO or Cos cell lines) encoding the polypeptides described in any preceding concept, and tetramers as defined in any preceding concept. ,mixture.

Optionally, the mixture is contained in a sterile container.
33. 33. The mixture of concept 32, wherein said cell line is in a medium comprising a secretory product of said cells, said secretory product comprising said tetramer.
34. The mixture of Concept 33, wherein said secreted product does not comprise monomers and/or dimers as defined in any one of Concepts 1-31.
35. 34. The mixture of concept 33, wherein said secreted product comprises an amount of said tetramer that is at least 10 times the amount of monomer and/or dimer.
36. 34. The mixture of concept 33, wherein said secreted product comprises said tetramer in a tetramer:monomer and/or dimer ratio of at least 90:10.
37. Yield of tetramers of protein effector domains (e.g., antibody variable domains or binding sites), including expressing tetramers of the polypeptide from cell lines (e.g., mammalian cells, CHO, HEK293 or Cos cell lines) and wherein said polypeptide is as defined in any preceding concept and comprises one or more effector domains, and said method optionally comprises said expressed isolating the tetramer.

Homologs, orthologs or equivalents have a tetramerization function.

Homolog: A gene, nucleotide or protein sequence that is related to a second gene, nucleotide or protein sequence by originating from a common ancestral DNA or protein sequence. The term homolog may be applied to the relationship between genes separated by the event of gene duplication or between genes separated by the event of gene duplication.

Orthologs: Orthologs are gene, nucleotide or protein sequences in different species that have evolved from a common ancestral gene, nucleotide or protein sequence by speciation. Orthologs usually retain the same function over the course of evolution.

In one example, a TD, ortholog or homolog is a TD of any one of proteins 1-119 listed in Table 2. In one example, an ortholog or homologue is a TD ortholog or homologue of any one of proteins 1-119 listed in Table 2. In one embodiment, instead of using the p53 tetramerization domain (p53-TD) or homologues or orthologs thereof, all aspects of the invention herein use proteins 1-119 listed in Table 2. Any one TD or its homologues or orthologs can be read as relating to use in or inclusion in a polypeptide, monomer, dimer, trimer or tetramer. The TD can be a TD of NHR2 (eg, human NHR2) or an ortholog or homologue thereof. The TD can be a TD of p63 (eg, human p63) or an ortholog or homologue thereof. The TD can be a TD of p73 (eg, human p73) or an ortholog or homologue thereof. This may have one or more of the following advantages:
- secretion of tetramers from mammalian or other eukaryotic cells, for example mammalian cells disclosed herein such as CHO, HEK293 or Cos;
- increased yield of secreted tetramer relative to monomer;
- increased yield of secreted tetramer over dimer;
- increased yield of secreted tetramers relative to trimers;
- increased yield of secreted tetramer relative to monomer and dimer combined;
- increased yield of secreted tetramer, relative to monomers, dimers and trimers combined;
- increasing the affinity or avidity of antigen binding in the tetramer containing the antigen binding site;
- Enhanced production and/or expression of tetramers with a size of 200 or 160 or 150 kDa or less.
Rituxan™; Rituximab; Zenapax™; Daclizumab; Simlect™; Gemtuzumab; Campus™; Alemtuzumab; Zevalin™; Ibritumomab; Humira™; Adalimumab; Erbitux(TM); Cetuximab; Avastin(TM); Bevacizumab; Tysabri(TM); Natalizumab; Actemra(TM); Cimzia™; Certolizumab; Simponi™; Golimumab; Ilaris™; Canakinumab; Motavizumab; Abslax™; Laxivacumab; Benlysta™; Belimumab; Yervoy™; Ipilimumab; ADCETRIS™; Kadcyla™; Ad-trastuzumab; Gazyva™ and obinutuzumab. Alternatively (e.g., for treating or preventing cancer in humans), each polypeptide or monomer may be ipilimumab (or Yervoy(TM)), tremelimumab, nivolumab (or Opdivo(TM)), pembrolizumab ( or Keytruda™), pidilizumab, BMS-936559, durvalumab and atezolizumab.

In one example, the tetramer is from the group consisting of ipilimumab (or Yervoy™), tremelimumab, nivolumab (or Opdivo™), pembrolizumab (or Keytruda™), pidilizumab, BMS-936559, durvalumab and atezolizumab 4 copies of the antigen binding site of a first antibody selected and optionally 4 copies of the antigen binding site of a second antibody selected from said group, wherein the first antibody and the second antibody Antibodies are different. For example, the first antibody is ipilimumab (or Yervoy™) and optionally the second antibody is nivolumab (or Opdivo™) or pembrolizumab (or Keytruda™). It is useful for treating or preventing cancer in humans.

In one example, a tetramer contains four copies of the antigen-binding site of Avastin. In one example, the tetramer contains four copies of the antigen binding site of Humira. In one example, the tetramer contains four copies of the Erbitux antigen binding site. In one example, the tetramer contains four copies of the Actemra™ antigen binding site. In one example, the tetramer contains four copies of the antigen binding site of sarilumab. In one example, the tetramer contains four copies of the dupilumab antigen binding site. In one example, the tetramer contains four copies of the antigen binding site of alirocumab or evolocumab. In one example, a tetramer contains four copies of the antigen binding site of . In one example, the tetramer contains four copies of the antigen-binding site of Remicade. In one example, the tetramer contains four copies of the Lucentis antigen binding site. In one example, the tetramer contains four copies of the Eylea™ antigen binding site. Such tetramers are useful for administration to humans to treat or prevent cancer. Such tetramers are useful for administration to humans to treat or prevent an ocular condition (e.g., wet AMD or diabetic retinopathy, e.g., when the binding site is that of Avastin, Lucentis or Eylea). is. Such tetramers are useful for administration to humans to treat or prevent angiogenesis.

In one example, a tetramer contains four copies of insulin. In one example, a tetramer contains four copies of GLP-1. In one example, a tetramer contains four copies of GIP. In one example, a tetramer contains four copies of exendin-4. In one example, a tetramer contains 4 copies of insulin and 4 copies of GLP-1. In one example, a tetramer contains 4 copies of insulin and 4 copies of GIP. In one example, a tetramer contains four copies of insulin and four copies of exendin-4. In one example, a tetramer contains four copies of GLP-1 and four copies of exendin-4. Such tetramers are useful for administration to humans to treat or prevent diabetes (eg, type II diabetes) or obesity.

Diseases and Conditions Monomers or multimers (e.g., dimers, trimers, tetramers or octamers) of the invention can be administered to a human or animal subject to treat or prevent a disease or condition in the subject. can be used in a method for

Optionally, the disease or condition is selected from:
(a) a neurodegenerative disease or condition;
(b) a brain disease or condition;
(c) a CNS disease or condition;
(d) memory loss or impairment;
(e) a heart or cardiovascular disease or condition, such as heart attack, stroke or atrial fibrillation;
(f) a liver disease or condition;
(g) a kidney disease or condition, such as chronic kidney disease (CKD);
(h) a pancreatic disease or condition;
(i) a pulmonary disease or condition, such as cystic fibrosis or COPD;
(j) a gastrointestinal disease or condition;
(k) diseases or conditions of the throat or oral cavity;
(l) an eye disease or condition;
(m) reproductive diseases or conditions, such as diseases or conditions of the vagina, labia, penis or scrotum;
(n) a sexually transmitted disease or condition, such as gonorrhea, HIV infection, syphilis or chlamydia infection;
(o) an ear disease or condition;
(p) a skin disease or condition;
(q) a heart disease or condition;
(r) nasal diseases or conditions; (s) blood diseases or conditions, such as anemia, such as anemia of chronic disease or cancer;
(t) viral infections;
(u) pathogenic bacterial infections;
(v) cancer;
(w) an autoimmune disease or condition, such as SLE;
(x) an inflammatory disease or condition such as rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease or IBD;
(y) autism;
(z) ADHD;
(aa) bipolar disorder;
(bb) ALS [amyotrophic lateral sclerosis];
(cc) osteoarthritis;
(dd) a congenital or developmental defect or condition;
(ee) miscarriage;
(ff) blood clotting status;
(gg) bronchitis;
(hh) dry or wet AMD;
(ii) angiogenesis (e.g., tumoral or intraocular);
(jj) common cold;
(kk) epilepsy;
(ll) fibrosis, such as liver or lung fibrosis;
(mm) fungal diseases or conditions, such as thrush;
(nn) Metabolic diseases or conditions such as obesity, anorexia, diabetes, type I or type II diabetes.
(oo) ulcers, such as gastric or skin ulcers;
(pp) dry skin;
(qq) Sjögren's Syndrome;
(rr) cytokine storm;
(ss) deafness, deafness or impairment;
(tt) slow or fast metabolism (i.e. slower or faster than average for the subject's weight, sex and age)
(uu) pregnancy disorders such as infertility or low fertility;
(vv) jaundice;
(ww) skin rashes;
(xx) Kawasaki disease;
(yy) Lyme disease;
(zz) allergies such as nut, grass, pollen, dust mite, cat or dog fur or dander allergies;
(aaa) malaria, typhoid, tuberculosis or cholera;
(bbb) depression;
(ccc) mental retardation;
(ddd) microcephaly;
(eee) malnutrition;
(fff) conjunctivitis;
(ggg) pneumonia;
(hhh) pulmonary embolism;
(iii) pulmonary hypertension;
(jjj) bone disorders;
(kkk) sepsis or septic shock;
(lll) sinusitis (Sinusitus);
(mmm) stress (e.g. industrial stress);
(nnn) thalassemia, anemia, von Willebrand's disease, or hemophilia;
(ooo) shingles or herpes labialis;
(ppp) menstruation;
(qqq) Low sperm count.

Neurodegenerative or CNS Diseases or Conditions for Treatment or Prevention In one example, the neurodegenerative or CNS disease or condition is Alzheimer's disease, senile psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinson's syndrome , Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinson's syndrome.

In one example, when the methods of the invention are practiced in a human or animal subject to treat a CNS or neurodegenerative disease or condition, the methods cause downregulation of Treg cells in the subject, thereby reducing systemic monocyte Encourage the derived macrophages and/or Treg cells to cross the choroid plexus and enter the subject's brain, thereby treating, preventing, or reducing progression of a disease or condition (eg, Alzheimer's disease). In one embodiment, the method causes an increase in IFN-gamma in the subject's CNS system (eg, in the brain and/or CSF). In one example, the method restores nerve fibers and/or reduces progression of nerve fiber damage. In one example, the method restores nerve myelin and/or reduces progression of nerve myelin damage. In one example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method comprises WO2015136541 (the disclosure of this document is, for example, Such methods, diseases, conditions, and potential therapeutic agents that can be administered to a subject to effect treatment and/or prevention of CNS and neurodegenerative diseases and conditions, e.g., agents such as immune checkpoint inhibitors, e.g. is incorporated herein by reference in its entirety to provide disclosure of the anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed in that document. can be used with the method. .

Cancers for Treatment or Prevention Cancers that may be treated include neovascularized tumors as well as non-neovascularized or substantially non-neovascularized tumors. Cancer can include non-solid tumors (hematological tumors such as leukemia and lymphoma) or can include solid tumors. Cancer types to be treated using the present invention include carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcoma, carcinoma, and melanoma. Also included are adult tumors/cancers and pediatric tumors/cancers.

Hematologic cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include acute leukemia (acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia and myeloblastic, promyeiocytic, myelomonocytic, monocytic). leukemia, including chronic leukemia (including chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myeiodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that do not normally contain cysts or fluid compartments. Solid tumors can be benign or malignant. Different types of solid tumors are named by the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma tumor, colon cancer, lymphoid tumor, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid Medullary carcinoma, papillary thyroid carcinoma, pheochromocytoma Sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, Testicular tumors, seminiferoma, bladder cancer, melanoma, and CNS tumors including glioma (including brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme) tumor, CNS lymphoma, germinoma, medu!loblastoma, schwannoma, craniopharyngioma, ependymoma, pinaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma , menangioma, neuroblastoma, retinoblastoma and brain metastasis).

Autoimmune disease acute disseminated encephalomyelitis (ADEM) for treatment or prevention
Acute necrotic hemorrhagic leukoencephalitis Addison's disease Agammaglobulinemia Alopecia areata Amyloidosis Ankylosing spondylitis Anti-GBM/anti-TBM nephritis Antiphospholipid syndrome (APS)
・Autoimmune angioedema ・Autoimmune aplastic anemia ・Autoimmune dysautonomia ・Autoimmune hepatitis ・Autoimmune hyperlipidemia ・Autoimmune immunodeficiency ・Autoimmune inner ear disease (AIED) )
・Autoimmune myocarditis ・Autoimmune oophoritis ・Autoimmune pancreatitis ・Autoimmune retinopathy ・Autoimmune thrombocytopenic purpura (ATP)
・Autoimmune thyroid disease ・Autoimmune urticaria ・Axonal neuropathy ・Barrow disease ・Behcet disease ・Bullous pemphigoid ・Cardiomyopathy ・Castleman disease ・Celiac disease ・Chagas disease ・Chronic fatigue syndrome ・Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)
- Chronic recurrent multifocal ostomyelitis (CRMO)
・Churg-Strauss syndrome ・Cicatricial pemphigoid/benign mucosal pemphigoid ・Crohn's disease ・Cogans syndrome
・Cold agglutinin disease ・Congenital heart block ・Coxsackie myocarditis ・Crest disease ・Essential mixed cryoglobulinemia ・Demyelinating neuropathy ・Dermatitis herpetiformis ・Dermatomyositis ・Devic's disease (neuromyelitis optica)
Discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrous alveolitis Giant cell arteritis (temporal arteritis)
・Giant cell myocarditis ・Glomerulonephritis ・Goodpasture syndrome ・Granulomatosis with polyangiitis (GPA)
・Graves' disease ・Guillain-Barré syndrome ・Hashimoto's encephalitis ・Hashimoto's thyroiditis ・Hemolytic anemia
・IgA nephropathy ・IgG4-related sclerosing disease ・Immunomodulatory lipoproteins ・Inclusion body myositis ・Interstitial cystitis ・Juvenile arthritis ・Juvenile diabetes (type 1 diabetes)
- Juvenile myositis - Kawasaki syndrome - Lambert-Eaton syndrome - Leukocytoclastic vasculitis - Lichen planus - Lichen sclerosus - Woody conjunctivitis - Linear IgA disease (LAD)
・Lupus (SLE)
Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD)
・Mohren's ulcer ・Much-Habermann disease ・Multiple sclerosis ・Myasthenia gravis ・Myositis ・Narcolepsy ・Neuromyelitis optica (Devic)
・Neutropenia ・Ocular cicatricial pemphigoid ・Optic neuritis ・Relapsing rheumatoid arthritis ・PANDAS (pediatric streptococcal autoimmune neuropsychiatric disorder)
・ Paraneoplastic cerebellar degeneration ・ Paroxysmal nocturnal hemoglobinuria (PNH)
Parry-Romberg syndrome Parsonnage-Turner syndrome Pars planitis (peripheral uveitis)
Pemphigus Perivenous neuropathy Perivenous encephalomyelitis Pernicious anemia POEMS syndrome Polyarteritis nodosa Type I, II and III autoimmune polyglandular syndrome Polymyalgia rheumatoid disease, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pus gangrenous Dermatosis, aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, recurrent polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE)
・Suzak syndrome ・Sympathetic ophthalmia ・Takayasu arteritis ・Temporal arteritis/giant cell arteritis ・Thrombocytopenic purpura (TTP)
・Tolosa-Hunt syndrome ・Transverse myelitis ・Type 1 diabetes ・Ulcerative colitis ・Undifferentiated connective tissue disease (UCTD)
・Uveitis ・Vasculitis ・Bullous skin disease ・Vitiligo ・Wegener's granulomatosis

Inflammatory diseases, Alzheimer's, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) for treatment or prevention
・Asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS)
・Systemic lupus erythematosus (SLE)
・Nephritis ・Parkinson's disease ・Ulcerative colitis

Polyvalent soluble TCR
This construct relates to a multivalent soluble TCR protein. In one aspect, the invention relates to soluble tetravalent and octavalent TCR analogues. The TCR proteins of the invention are capable of self-assembly from monomers and are fully human in origin. The protein is a multimer containing the ETO NHR2 multimerization domain. The invention also relates to methods of constructing multimeric soluble TCRs and methods of using such proteins.

Attempts to exploit alternative soluble TCR formats as therapeutic molecules have lagged far behind the plethora of antibody formats. This is due in large part to the inherent problem of solubility of the TCR heterodimeric transmembrane proteins when the extracellular TCR α/β chains are separated from the transmembrane and cytoplasmic domains. Second, the inherent low affinity and avidity of these molecules for their cognate ligands at target sites has greatly hampered their development as therapeutic molecules.

To overcome these drawbacks, this configuration of the invention provides TCR proteins that are both multivalent and soluble. Multivalency increases the TCR's avidity for allogeneic pMHC, and solubility allows the use of the TCR outside the transmembrane environment. Therefore, in the first aspect,
(i) a TCR extracellular domain;
A multivalent heterodimeric soluble T cell receptor capable of binding to pMHC complexes is provided comprising (ii) (ii) an immunoglobulin constant domain and (iii) (iii) the NHR2 multimerization domain of ETO.

The use of Ig constant domains provided TCR extracellular domains with stability and solubility, and multimerization via the NHR2 domain provided multivalency and increased avidity. Advantageously, all domains are of human origin or are based on human protein sequences.

By using Ig constant domains to stabilize and make the TCR soluble, the use of non-natural disulfide bonds is avoided. Advantageously, therefore, the TCRs of the present invention do not contain non-natural disulfide bonds.

In one embodiment, said complex comprises a heavy chain and a light chain, and each light chain comprises a TCR Vα domain and an immunoglobulin C _α domain, and each heavy chain comprises a TCR Vβ domain and an immunoglobulin CH1 domain. include.

In one embodiment, each light chain additionally comprises a TCR Ca domain and each heavy chain additionally comprises a TCR Vβ domain.

In embodiments, the TCR and immunoglobulin domain may be separated by a flexible linker.

The NHR2 multimerization domain is advantageously attached to the C-terminus of the immunoglobulin domain. Thus, each heavy and light chain dimer will be attached to one multimerization domain, and the heavy-light chain dimers will associate into multivalent oligomers.

In embodiments, the multimerization domain and the immunoglobulin domain are separated by a flexible linker. In certain embodiments, this allows the multimerization domain to multimerize without interference from the immunoglobulin domain.

In embodiments, the TCR protein may further comprise an immunoglobulin hinge domain. The hinge domain allows dimerization of heavy-light chain dimers, which allows further multimerization of TCR proteins. For example, multimerization domains that form tetramers can use immunoglobulin hinge domains to form multimers up to octamers. Similarly, dimerizing multimerization domains can form tetramers in the presence of a hinge domain.

In embodiments, the TCR proteins of the invention are tetravalent.

In embodiments, the TCR proteins of the invention are octavalent.

The present invention provides soluble TCRs that stably assemble in a tetravalent heterodimeric format using the nerve homology region 2 (NHR2) domain found in ETO family proteins in humans (Liu et al. 2006). . NHR2 domains have been found to naturally form homotetramers formed from the pairing of two NHR2 homodimers. NHR2 operably linked to extracellular TCRa or TCRβ chains preferentially forms tetravalent heterodimeric soluble TCR protein molecules that self-assemble sequentially from monomers and then homodimers. (Fig. 1).

TCR proteins that assemble into octamers can be generated using NHR2 domains by using immunoglobulin hinge domains.

In a further aspect, the TCR proteins of the invention can be linked to biologically active polypeptides/effector molecules. Examples of such polypeptides can include immunologically active moieties such as cytokines, binding proteins such as antibodies or targeting polypeptides, and the like.

The present invention provides methods for making tetravalent and octavalent heterodimeric soluble TCRs, DNA vectors encoding the proteins that can be used to transfect host cells of interest, and novel highly sensitive multivalent It further relates to the use of soluble soluble TCR protein molecules. Applications for use include, but are not limited to, therapeutics, diagnostics and drug discovery.

In a further aspect, the invention provides methods of constructing multivalent immunoglobulin molecules in an efficient manner without the use of non-human construct components.

therefore,
A multimeric immunoglobulin is provided that includes (i) an immunoglobulin variable domain, and (ii) an NHR2 multimerization domain of ETO.

The immunoglobulin variable domains are preferably antibody variable domains. Such domains are fused to ETO NHR2 multimerization domains, which provide a means for forming tetramers of immunoglobulin variable domains.

The ETO NHR2 domain is more efficient than p53 and similar multimerization domains in producing immunoglobulin multimers, allowing the production of multimeric immunoglobulin molecules without the use of non-human components in the construct.

Also provided is a method of producing a multimeric immunoglobulin comprising expressing an immunoglobulin variable domain in fusion with the NHR2 domain of ETO and assembling the variable domains into a multimer.

Preferably, immunoglobulin variable domains are attached to one or more immunoglobulin constant domains.

Advantageously, the immunoglobulin domain is an antibody domain. For example, variable domains can be VH and VL antibody domains. For example, the constant domain is an antibody CH1 domain.

In one embodiment, multimeric immunoglobulin molecules according to the invention, both TCR and non-TCR immunoglobulins, are produced for screening by phage display or another display technology. Thus, for example, multivalent immunoglobulins are produced as fusions with phage coat proteins. For each immunoglobulin produced fused to a coat protein, other immunoglobulin molecules are produced without the coat protein so that they can assemble on the phage surface as a result of NHR2 multimerization.

This aspect of the invention, detailed above, relates to nucleic acid sequences and methods for producing novel multivalent, eg, tetravalent and octavalent, soluble proteins. In one aspect, among others, the soluble protein is a TCR assembled in a tetravalent heterodimeric format capable of binding four pMHCs with high sensitivity, affinity and specificity. Soluble tetravalent heterodimeric TCRs are unique protein molecules composed of either whole or partial extracellular TCR α/β chains. Extracellular TCR α/β chains are linked to immunoglobulin CH1 and CL (either CK or CX) domains. This ligation allows stable formation of the heterodimeric TCR α/β. A unique feature in the context of soluble tetravalent TCRs is the _NHR2 homotetrameric domain of the ETO family of proteins operably linked to the _C -terminus of CH1 or CL. Ligation of the NHR2 domain to the heterodimeric a/pTCR in this manner allows it to self-assemble into a tetravalent format within the cell and be subsequently secreted into the supernatant as a soluble protein. .

TCR Extracellular Domain The TCR extracellular domain is composed of variable and constant regions. These domains exist in T-cell receptors much as they exist in antibodies and other immunoglobulin domains. The TCR repertoire has extensive diversity created by the same gene rearrangement machinery used in antibody heavy and light chain genes (Tonegawa, S. (1988) Biosci. Rep. 8:3-26). Most of the diversity is generated at the junction of the variable (V) and joining (or diversity, D) regions encoding the complementarity determining regions 3 (CDR3) of the a and β chains (Davis and Bjorkman ( 1988) Nature 334:395-402). Databases of TCR genes are available, such as the IMGT LIGM database, and methods for cloning TCRs are known in the art. For example, Bentley and Mariuzza (1996) Ann. Rev. Immunol. 14:563-590; Moysey et al., Anal Biochem. 2004 Mar 15;326(2):284-6; Walchli, et al. Practical Approach to T-Cell Receptor Cloning and Expression. See PLoS ONE 6(11): e27930.

Immunoglobulin variable domains Antibody variable domains are known in the art and are available from a variety of sources. Databases of antibody variable domain sequences exist, such as IMGT and Kabat, and variable domains can be produced by cloning and expression of native sequences, or by synthetic nucleic acid synthesis, according to established techniques.

Methods for constructing bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al. (1991) J Immunol., 147: 3610; Breitling et al. (1991) supra; Barbas et al. (1992) supra; Hawkins and Winter (1992) J Immunol., 22: 867; Marks et al., 1992, J Bioi. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313; incorporated herein by reference).

One particularly advantageous approach is the use of scFv phage libraries (Huston et al., 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991) Nature, 352: 624; Marks et al. Chiswell et al. (1992) Trends Biotech., 10:80; Marks et al. (1992) J Bioi. Chem., 267). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Precision of the phage display approach, for example, as described in WO96/06213 and WO92/01047 (Medical Research Council et al.) and WO97/08320 (Morphosys), which are incorporated herein by reference. Chemicals are also known.

Such techniques can be adapted for the production of multimeric immunoglobulins by fusion of NHR2 multimerization domains to antibody variable domains.

Immunoglobulin constant domains The immunoglobulin constant domains referred to herein are preferably antibody constant domains. The constant domains vary in sequence between antibody subtypes, preferably the constant domains are IgG constant domains. Preferably, the constant domain is the CH1 constant domain. Antibody constant domains are well known in the art and are available from numerous sources and databases, such as the IMGT and Kabat databases.

Fusions of antibody constant domains to immunoglobulin variable domains are also known in the art, eg, in the construction of engineered Fab antibody fragments.

A linker flexible linker can be used to connect the TCR variable domain-Ig constant domain to the NHR2 multimerization domain. This allows the TCR and multimerization domains to function without steric hindrance from each other or from other molecules in the multimeric complex. Suitable linkers include, for example, glycine repeats, glycine-alanine repeats, Gly(4)Ser linkers, or flexible polypeptide linkers as described in Reddy Chichili et al., 2012 Protein Science 22:153-167.

An immunoglobulin hinge domain , an Ig hinge domain, preferably an antibody hinge domain in the present invention, is a domain that joins antibody constant regions in natural antibodies. This domain thus provides for the natural dimerization of molecules containing antibody constant domains. It is present, for example, in whole antibodies, such as IgG, in addition to F(ab)2 antibody fragments. This region contains two natural interchain disulfide bonds that connect the two CH1 constant domains together.

In one embodiment, the multimerization domain may be attached to an Ig constant domain or hinge domain. When the hinge domain is present, the multimerization domain forms a TRC octamer comprising four dimers of TCR variable-Ig constant domains joined at the hinge region. In the absence of the hinge region, the multimerization domain leads to tetramer formation. Preferably, the multimerization domain is attached to the C-terminus of the constant domain or hinge region.

Biologically Active Molecules One or more biologically active molecules or effector molecules (EM) can be attached to the multimers, eg, multimeric TCR proteins, of the invention. Such a molecule can be, for example, an antibody, such as an anti-CD3 antibody or antibody fragment, particularly an antibody capable of assisting TCR immune recognition and function.

In some aspects, the bioactive molecule can be a bioactive molecule such as a cytotoxic drug, toxin, or cytokine, as described in more detail below. Examples of bioactive molecules include chemokines such as MIP-1b, cytokines such as IL-2, growth factors such as GM-CSF or G-CSF, toxins such as ricin, cytotoxic agents such as doxorubicin or taxanes, radioactive and Examples include labels such as fluorescent labels. See US Patent Application Publication No. 20110071919 for examples of bioactive molecules that can be conjugated to TCRs.

In other aspects, the bioactive molecule is selected, for example, from the group consisting of: a group capable of binding to a molecule that extends the half-life of the polypeptide ligand in vivo; A molecule that extends. Such a molecule may be, for example, HSA or a cell matrix protein, and the group capable of binding to a molecule that extends the half-life of a TCR molecule in vivo is an antibody or antibody fragment specific for HSA or a cell matrix protein. be.

In one embodiment, the bioactive molecule is a binding molecule, such as an antibody fragment. Two, three, four, five or more antibody fragments can be linked together using suitable linkers. The specificity of any two or more of these antibody fragments may be the same or different, and if they are the same, the specificity of any two or more of these antibody fragments may be the same, and if they are the same, the specificity of any two or more of these antibody fragments may be the same, and if they are the same, the Multivalent binding structures with increased avidity are formed.

A bioactive molecule may also be an effector group, such as an antibody Fc region.

Attachment to the N-terminus or C-terminus may be performed prior to or after assembly of the TCR molecules or engineered polypeptides into multimers. Thus, TCR fusions with Ig constant domains may be produced (synthetically or by expression of nucleic acids) in which an N-terminal or C-terminal bioactive molecule is already present. However, in certain aspects, N-terminal or C-terminal additions are made after the TCR fusion is made. For example, fluorenylmethyloxycarbonyl chloride can be used to introduce an Fmoc protecting group at the N-terminus of the TCR fusion. Fmoc binds serum albumins such as HSA with high affinity, and Fmoc-Trp or FMOC-Lys bind with increased affinity. After synthesizing the peptide with the Fmoc protecting group attached, it can be linked to the scaffold through the cysteine. An alternative is a palmitoyl moiety that also binds to HSA, which has been used, for example, in liraglutide to extend the half-life of this GLP-1 analogue.

Alternatively, a TCR fusion (TCR fusinon) can be modified at the N-terminus, eg, using an amine- and sulfhydryl-reactive linker Ne-maleimidocaproyloxy)succinimide ester (EMCS). Via this linker, the TCR can be linked to other polypeptides, such as antibody Fc fragments.

NHR2 domain AML1/ETO is a fusion protein resulting from t(8;21) found in acute myeloid leukemia (AML) of the M2 subtype. AML1/ETO contains the N-terminal 177 amino acids of RUNX1 fused in-frame with most of ETO (575 aa). ETO nerve homology domain 2 is responsible for many of the AML1/ETO-associated biological activities, such as oligomerization and protein-protein interactions. This domain has been characterized in detail in Liu et al (2006). See GenBank Accession No. NG_023272.2.

In one aspect of the invention, the protein assembled in a soluble, multivalent format is a TCR partially or wholly composed of the extracellular domains of the TCR a-chain and β-chain. The TCR a and β chains are stabilized by immunoglobulin C _H 1 and C _L domains and can be organized in the following configurations:
1. Vα-C _L and VβC _H 1
2. Vα-C _H 1 and Vβ-C _L
3. VαCα-C _L and VβCβ-C _H 1
4. VαCαC _H 1 and VβCβC _L

In one aspect of the invention, the extracellular TCR domain is linked to immunoglobulin CH1 and CL domains via an optionally present peptide linker (L) that promotes protein flexibility and promotes optimal protein folding. be.
1. Vα-(L)-C _L and Vβ-(L)-C _H 1
2. Vα-(L)-C _H 1 and Vβ-(L)-C _L
3. VαCα-(L)-C _L and VβCβ-(L)-C _H 1
4. VαCα-(L)-C _H 1 and VβCβ-(L)-C _L

In another aspect of the invention, the tetramerization domain (TD), such as the NHR2 homotetramer domain, is the C of either the immunoglobulin CH1 or CL domain linked to the extracellular TCR a and β chains. ligated to the end. The NHR2 domain may optionally be linked to the CH1 or CL domain via a peptide linker. The resulting tetravalent heterodimeric TCR protein can be assembled with the following configuration ((L) is an optional peptide linker):
1. Vα-(L)-C _L and Vβ-(L)-C _H 1-(L)-TD
2. Vα-(L)-C _H 1-(L)-TD and Vβ-(L)-C _L
3. VαCα-(L)-C _L and VβCβ-(L)-C _H 1-(L)-TD
4. VαCα-(L)-C _H 1-(L)-TD and VβCβ-(L)-C _L
5. Vα-(L)-C _L -(L)-TD and Vβ-(L)-C _H 1
6. Vα-(L)-C _H 1 and Vβ-(L)-C _L -(L)-TD
7. VαCα-(L)-C _L -(L)-TD and VβCβ-(L)-C _H 1
8. VαCα-(L)-C _H 1 and VβCβ-(L)-C _L -(L)-TD

The sensitivity of a soluble TCR to its cognate pMHC can be enhanced by increasing the avidity effect. This is achieved by increasing the number of antigen binding sites facilitated by the tetramerization domain. And this also increases the molecular weight of the protein molecule compared to the monovalent soluble TCR, thus prolonging serum retention in circulation. Increased serum half-lives also increase the likelihood that these molecules will interact with their cognate target antigens.

A tetravalent heterodimeric soluble TCR protein molecule can simultaneously bind to 1, 2, 3 or 4 pMHC displayed on a single cell or present its cognate pMHC1 Binds to 1, 2, 3 or 4 different cells simultaneously.

The TCR α and β chain sequences used in the present invention may be from known TCRs specific for a particular pMHC or may be obtained using techniques known in the art such as phage display. may be identified de novo by screening using Furthermore, TCR sequences are not limited to α and β chains in the present invention, TCR8 and TCR8 cloned directly from human T cells or identified by directed evolution using recombinant DNA technology. γ or ε chains and sequence variations thereof can also be incorporated.

In another aspect of the invention, tetravalent heterodimeric soluble TCR protein molecules are preferentially produced in mammalian cells for optimal production of soluble, stably and correctly folded protein molecules.

Multimeric (eg, tetrameric or octameric) or multivalent TCRs according to the invention can be expressed in cells, such as mammalian cells, using any suitable vector system. The pTT5 expression vector is one example of an expression system used to express multivalent soluble TCRs. The pTT5 expression system allows the transient production of high levels of recombinant protein in suspension-adapted HEK293 EBNA cells (Zhang et al. 2009). It is an origin of replication recognized by the viral protein Epstein-Barr nuclear antigen 1 (EBNA-1), which mediates episomal replication of DNA plasmids in conjunction with host cell replication factors to enable enhanced expression of recombinant proteins. oriP). Other suitable vector systems for mammalian cell expression that are known in the art and commercially available can be used in the present invention.

Tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced by transiently expressing the gene from an expression vector.

In another embodiment, tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced from engineered stable cell lines. Cell lines can be engineered to produce protein molecules using genome engineering techniques known in the art that integrate genes encoding protein molecules into the genome of the host cell as single or multiple copies. The site of DNA integration can be a defined location within the host genome or can be randomly integrated to obtain maximal expression of the desired protein molecule. Genome engineering techniques include homologous recombination, transposon-mediated gene transfer such as the PiggyBac transposon system, site-specific recombinases such as recombinase-mediated cassette exchange, endonucleases such as CRISPR/Cas9, TALENs, zinc finger nucleases and meganucleases. These include, but are not limited to, nuclease-mediated gene targeting, virus-mediated gene transfer such as lentivirus.

In another aspect of the invention, the tetravalent heterodimeric soluble TCR protein molecules or other multimers are also isolated from E. coli as inclusion bodies. produced by overexpression in the cytoplasm of E. coli and refolded in vitro after purification by affinity chromatography to produce functional protein molecules capable of properly binding its cognate pMHC or antigen.

In another aspect of the invention, expression of tetravalent heterodimeric soluble TCR protein molecules or other multimers is not limited to mammalian or bacterial cells, but includes insect cells, plant cells, yeast cells, and the like. It can also be expressed and manufactured in eukaryotic cells.

In another aspect of the invention, heterodimeric soluble TCR molecules or other multimers are produced as octavalent protein complexes, e.g., with up to eight binding sites for their cognate pMHC ( Figure 2). Multiple antigen binding sites allow the molecule to bind up to 8 pMHC displayed on one cell or to pMHC displayed on up to 8 different cells. , thus producing a highly sensitive soluble TCR.

The heterodimeric soluble TCR portion of the molecule is made a bivalent molecule by fusing an immunoglobulin hinge domain to the C-terminus of either the CH1 or CL domains which themselves are linked to either the TCR a-chain or the β-chain. It is said that The hinge domain allows the connection of the two heavy chains to give a structure similar to IgG. A tetramerization domain such as NHR2 is linked to the C-terminus of the hinge domain via an optional peptide linker. Attaching immunoglobulin hinges to the C- and N-termini of the Ig CH1 or CL and NHR2 domains, respectively, allows assembly of two NHR2 monomers, termed monomer ² . In this configuration, unless a long flexible linker is provided between the hinge and the NHR2 domain, the two NHR2 domains most likely do not form homodimers by antiparallel association due to structural constraints. is expected. Tetramerization and hinge domain ligation to heterodimeric soluble TCRs via immunoglobulin CH1 or CL domains initiates stepwise self-assembly of octavalent soluble TCRs formed through NHR2 homotetramer ² make it possible. Self-assembly of the octavalent soluble TCR is via an intermediate protein complex of NHR2 monomer ² and homodimer ² (Fig. 2). The resulting octavalent heterodimeric soluble TCR protein molecule has excellent sensitivity to its cognate pMHC, and thus TCR to its pMHC ligands with affinities well above those found in nature. offers the unique advantage of identifying unknown antigens or pMHCs without the need to affinity mature a . In particular, it is useful for identifying pMHC recognized by uncharacterized tumor-specific T cells and T cells involved in other diseases such as autoimmune diseases. A number of different configurations of octavalent heterodimeric soluble TCR protein molecules can be produced. Some examples are given below.
1. Vα-(L)-C _L and Vβ-(L)-C _H 1-hinge-(L)-TD
2. Vα-(L)-C _H 1-hinge-(L)-TD and Vβ-(L)-C _L
3. Vα-Cα-(L)-C _L and Vβ-Cβ-(L)-C _H 1-hinge-(L)-TD
4. VαCα(L)-C _H 1-hinge-(L)-TD and VβCβ-(L)-C _L
5. Vα-(L)-C _L -(L)-TD and Vβ-(L)-C _H 1-hinge6. Vα-(L)-C _H 1-hinge and Vβ-(L)-C _L -(L)-TD
7. Vα-Cα-(L)-C _L -(L)-TD and Vβ-Cβ(L)-C _H 1-hinge8. Vα-Cα-(L)-C _H 1-hinge and Vβ-Cβ-(L)-C _L -(L)-TD

In another aspect of the invention, self-assembled multivalent proteins, preferentially tetravalent and octavalent heterodimeric soluble TCRs, are fused or conjugated to bioactive agent/effector molecules, thereby These molecules are directed to desired cell populations, such as cancer cells, allowing them to specifically exert their therapeutic effect. The tumor-targeting ability of monoclonal antibodies that guide effector molecules such as cytotoxic drugs, toxins, or cytokines is well established (Perez et al. 2014; Young et al. 2014). Similarly, the multivalent soluble TCR molecules outlined in this invention can also be fused with effector proteins and polypeptides or conjugated to cytotoxic agents. Examples of effector protein molecules suitable for use as fusion proteins with the multivalent protein complexes outlined in the present invention include IFNa, IFNβ, IFNy, IL-2, IL-11, IL-13, granules globulocyte-colony stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor [TNF]a, IL-7, IL-10, IL-12, IL-15, IL-21 , CD40L, and TRAIL, co-stimulatory ligands are B7.1 or B7.2, chemokines DC-CK1, SDF-1, fractalkine, lyphotactin, IP-10, Mig, MCAF, MlP-la, MIP-1 /3, IL-8, NAP-2, PF-4, and RANTES or active fragments thereof. Examples of toxic agents suitable for use as fusion proteins or conjugated to multivalent protein complexes described in the present invention include diphtheria toxin, ricin, Pseudomonas exotoxin, and the like. Cytotoxic drugs such as, but not limited to, toxins, auristatins, maytansines, calicheamicins, anthracyclines, duocarmycins, pyrrolobenzodiazepines. The cytotoxic drug can be conjugated via a selectable linker, which is non-cleavable or cleavable by a protease, or acid-labile.

Cytotoxic drugs can be conjugated in a site-specific manner to remove heterogeneity and improve conjugate stability. This can be achieved by engineering specific cysteine residues or using enzymatic conjugation through glycosyltransferases and transglutaminases (Panowski et al. 2014).

In another aspect of the invention, the multivalent protein complex is covalently linked to a molecule that allows detection, such as a fluorescent agent, a radioactive agent or an electron transfer agent.

In another aspect of the invention, an effector molecule (EM) is fused to a multivalent protein complex via the C-terminus of a tetramerization domain such as NHR2 via an optionally present peptide linker. Fusions through the NHR2 domains can be arranged to produce multivalent protein complexes in many different configurations. Some examples of protein constructs that can be produced using a tetravalent heterodimeric soluble TCR are shown below:
1. Vα-(L)-C _L and Vβ-(L)-C _H 1-(L)-TD-(L)-EM
2. Vα-(L)-C _H 1-(L)-TD-(L)-EM and Vβ-(L)-C _L
3. Vα-Cα-(L)-C _L and Vβ-Cβ-(L)-C _H 1-(L)-TD-(L)-EM
4. Vα-Cα-(L)-C _H 1-(L)-TD-(L)-EM and Vβ-Cβ-(L)-C _L
5. Vα-(L)-C _L -(L)-TD-(L)-EM and Vβ-(L)-C _H 1
6. Vα-(L)-C _H 1 and Vβ-(L)-C _L -(L)-TD-(L)-EM
7. Vα-Cα-(L)-C _L -(L)-TD-(L)-EM and Vβ-Cβ-(L)-C _H 1
8. Vα-Cα-(L)-C _H 1 and Vβ-Cβ-(L)-C _L- (L)-TD-(L)-EM

In another aspect of the invention, effector molecules (EM) are fused to the multivalent protein complex at the C-terminus of either immunoglobulin CH1 or CL1 domains via an optionally present peptide linker. Fusions of EM via immunoglobulin domains can be arranged to produce multivalent protein complexes in many different configurations. Some examples of protein constructs that can be produced using a tetravalent heterodimeric soluble TCR are shown below:
9. Vα-(L)-C _L -(L)-EM and Vβ-(L)-C _H 1-(L)-TD
10. Vα-(L)-C _H 1-(L)-TD and Vβ-(L)-C _L- (L)-EM
11. Vα-Cα-(L)-C _L -(L)-EM and Vαβ-Cβ-(L)-C _H 1-(L)-TD
12. Vα-Cα-(L)-C _H 1-(L)-TD and Vβ-Cβ-(L)-C _L -(L)-EM
13. Vα-(L)-C _L -(L)-TD and Vβ-(L)-C _H 1-(L)-EM
14. Vα-(L)-C _H 1-(L)-EM and Vβ-(L)-C _L- (L)-TD
15. Vα-Cα-(L)-C _L -(L)-TD and Vβ-Cβ-(L)-C _H 1-(L)-EM
16. Vα-Cα-(L)-C _H 1-(L)-EM and Vβ-Cβ-(L)-C _L -(L)-TD

In another aspect of the invention, the effector molecule (EM) is at the C-terminus of either the immunoglobulin CH1 or CL1 domain via an optionally present peptide linker and also the tetramerization domain (e.g. NHR2) is fused at the C-terminus of the multivalent protein complex. This approach allows a fusion of two effector molecules to be fused by the TCR heterodimeric complex. The fusion of EM via an immunoglobulin domain and a tetramerization domain can be arranged to produce multivalent protein complexes in many different configurations. Some examples of protein constructs that can be produced using a tetravalent heterodimeric soluble TCR are shown below:
17. Vα-(L)-C _L -(L)-EM and Vβ-(L)-C _H 1-(L)-TD-(L)-EM
18. Vα-(L)-C _H 1-(L)-TD-(L)-EM and Vβ-(L)-C _L- (L)-EM
19. Vα-Cα-(L)-C _L -(L)-EM and VβCβ-(L)-C _H 1-(L)-TD-(L)-EM
20. Vα-Cα-(L)-C _H 1-(L)-TD-(L)-EM and Vβ-Cβ-(L)-C _L -(L)-EM
21. Vα-(L)-C _L -(L)-TD-(L)-EM and Vβ-(L)-C _H 1-(L)-EM
22. Vα-(L)-C _H 1-(L)-EM and Vβ-(L)-C _L- (L-)TD-(L)-EM
23. Vα-Cα-(L)-C _L -(L)-TD-(L)-EM and Vβ-Cβ-(L)-C _H 1-(L)-EM
24. Vα-(L)-C _H 1-(L)-EM and Vβ-Cβ-(L)-C _L -(L)-TD-(L)-EM

In another aspect of the invention, multivalent protein complexes are fused to protein tags that facilitate purification. Purification tags are known in the art and can include but are not limited to the following tags: His, GST, TEV, MBP, Strep, FLAG.

Non-TCR Multimers The present invention provides a unique method of assembling proteins into a soluble, multivalent format with the ability to bind multiple interacting domains or antigens. Proteins may be monomeric, homodimeric, heterodimeric or oligomeric, preferentially directly or indirectly involved in the immune system or having the ability to modulate an immune response. Examples include, but are not limited to, the TCR, peptides MHC class I and class II, antibodies or antigen binding portions thereof, and binding proteins with alternative non-antibody protein scaffolds.

In another aspect of the invention, the interacting domain or antigen is any cell surface expressed or secreted protein, peptides associated with MHC class I or II, or any protein associated with pathogens such as viral and bacterial proteins. possible.

Non-TCR multimers can be multimers of antibodies or antibody fragments, such as dAbs of Fabs. Examples of dAbs and Fabs according to the invention include:

Examples of multivalent DAbs25. VH-(L)-NHR2
26. VL(λ or κ)-(L)-NHR2
27. VH-(L)-NHR2-(L)-EM
28. VL(λ or κ)-(L)-NHR2-(L)-EM
29. VH-CH1-(L)-NHR2
30. VL(λ or κ)-CL-(L)-NHR2
31. VH-CH1-(L)-NHR2-(L)-EM
32. VL(λ or κ)-CL-(L)-NHR2-(L)-EM

Examples of multivalent Fabs33. VH-CH1-(L)-NHR2 and VL(λ or κ)-CL
34. VL(λ or κ)-CL-(L)-NHR2 and VH-CH1
35. VH-CH1-hinge-(L)-NHR2 and VL(λ or κ)-CL
36. VL (λ or κ)-CL-hinge-(L)-NHR2 and VH-CH1
37. VH-CH1-(L)-NHR2-(L)-EM and VL(λ or κ)-CL
38. VL(λ or κ)-CL-(L)-NHR2-(L)-EM and VH-CH1
39. VH-CH1-hinge-(L)-NHR2-(L)-EM and VL(λ or κ)-CL
40. VL(λ or κ)-CL-hinge-(L)-NHR2-(L)-EM and VH-CH1
41. VH-CH1-(L)-NHR2 and VL(λ or κ)-CL-(L)-EM
42. VL(λ or κ)-CL-(L)-NHR2 and VH-CH1-(L)-EM
43. VH-CH1-hinge-(L)-NHR2 and VL(λ or κ)-CL-(L)-EM
44. VL(λ or κ)-CL-hinge-(L)-NHR2 and VH-CH1-(L)-EM

In the above examples, (L) represents an optionally present peptide linker and EM represents a bioactive agent or effector molecule such as a toxin, drug or cytokine, including binding agents such as antibodies, Fabs and ScFvs. Contains molecules.

Variable light chains can be either Vλ or Vκ.

In one aspect of the invention, the assembled tetramerized protein molecules are, in one example, human pMHC for drug discovery applications using animal drug discovery platforms (e.g. mouse, rat, rabbit, chicken). obtain. In such contexts, the tetramerization domain is preferentially expressed and manufactured from a gene originating from the intended animal species. One example of such a drug discovery application would be the use of tetramerized human pMHC as an antigen for immunization in rats, for example. When rats are immunized with pMHC, the immune response is specifically directed against human pMHC rather than the tetramerization domain of the protein complex.

Multivalent antibodies can be prepared, for example, using single domain antibody sequences fused to NHR2 multimerization domains.

In aspects related to the present invention, the tetravalent protein can be a peptide used as a probe for molecular imaging of tumor antigens. The multivalent binding properties of such probes over monovalent molecular probes have enhanced affinities, avidities and retention times in vivo and this enhances tumor targeting in vivo. have unique advantages.

The multimerization domain is the NHR2 domain described above. Preferably, the polypeptide is stabilized and/or rendered soluble by use of an Ig constant domain fused to the polypeptide, the fusion providing a tetramer of the polypeptide. An Ig hinge domain can be used to provide an octamer.

Uses of Multimers Multimeric TCR proteins according to the invention are useful in any application where soluble TCR proteins are indicated. Particular advantages of the TCR proteins of the invention include increased avidity for selected targets and/or the ability to bind multiple targets.

Thus, in one aspect, the multivalent heterodimeric soluble TCR protein molecules of the invention can be used to selectively inhibit immune responses, eg, for suppression of autoimmune responses. The multivalent, eg, tetravalent, nature of these soluble protein molecules endows them with sharp sensitivity and binding affinity to compete with antigen-specific interactions between T cells and antigen-presenting cells. This type of neutralizing effect could be therapeutically beneficial in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, Graves' disease, vasculitis and type 1 diabetes.

Similarly, tetravalent heterodimeric soluble TCR protein molecules selectively inhibit T-cell recognition of specific transplantation and self-antigens that bind to target molecules, thus inhibiting cell-to-cell interactions and thereby inhibiting cell-to-cell interactions. It can be used to prevent transplant rejection.

In another aspect of the invention, tetravalent heterodimeric soluble TCR protein molecules are used in clinical studies such as toxicology, infectious disease studies, neurological studies, behavioral and cognitive studies, reproductive, genetics and xenograft studies. can be used in

Tetravalent heterodimeric soluble TCR protein molecules with enhanced sensitivity to allogeneic pMHC can be used for diagnostic purposes using biological samples obtained directly from human patients. The enhanced sensitivity of the tetravalent heterodimeric soluble TCR allows detection of potential disease-relevant peptides presented on MHC that are found to be naturally expressed at low densities. These molecules can also be used for patient stratification for patient enrollment in relevant clinical trials.

In another aspect of the invention, the octavalent heterodimeric soluble TCR protein molecules can be used in pharmaceutical preparations for the treatment of various diseases.

In another aspect related to the present invention, octavalent heterodimeric soluble TCR protein molecules can be used as probes for molecular imaging of tumors or prepared as therapeutic proteins.

Example 1 Generation of Tetravalent and Octavalent Soluble Heterodimeric NY-ESO-1 TCR Molecules We demonstrate a method to generate soluble heterodimeric TCR molecules that are valent and octavalent. These formats overcome problems associated with target site solubility and avidity for cognate ligands.

In order to exemplify the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR as stable and soluble molecules, a high-level Affinity TCR αβ variable sequences are used in this example.

The SLLMWITQC-HLA-A ^* 0201-specific high-affinity NY-ESO TCR αβ chains (composing TCR Vα-Ca and Vβ-Cp, respectively) used in this example have a signal peptide sequence (MGWSCIILFLVATATGVHS). are reported in International Publication No. 2005/113595. A histidine tag was incorporated at the C-terminus of the NHR2 domain to aid in protein purification.

DNA constructs encoding components of the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR were synthetically constructed as a two-vector system to allow soluble expression and functional assembly in mammalian cells. . A schematic representation of the two assembled TCR chains (a and β chains) from which the DNA sequences for cloning into the expression vector are synthesized is shown in FIGS. 3 and 4, and their amino acid sequences are shown in FIGS. 6.

The pTT5 expression system allows the transient production of high levels of recombinant protein in suspension-adapted HEK293 EBNA cells (Zhang et al. 2009). It is an origin of replication recognized by the viral protein Epstein-Barr nuclear antigen 1 (EBNA-1), which mediates episomal replication of DNA plasmids in conjunction with host cell replication factors to enable enhanced expression of recombinant proteins. oriP). Therefore, the pTT5 expression vector is chosen for cloning the components for the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR molecules.

A synthesized DNA fragment containing the TCR αβ chain is digested at the restriction site (RS) with a restriction enzyme (FastDigest, Fermantas) and the DNA separated on a 1% agarose gel. Excise the correct size DNA fragment and purify the DNA using Qiagen's gel extraction kit. The pTT5 vector is also digested with the same restriction enzymes and the linearized plasmid DNA is purified from the excised agarose gel. The digested TCR αβ chain was cloned into the digested pTT5 vector to generate four expression vectors (pTT5-ts-NY-ESO-1-TCRa, pTT5-ts-ESO-1-TCRβ, pTT5-os-NY- ESO-1-TCRa and pTT5-os-ESO-1-TCRβ) are obtained.

Expression of Tetravalent and Octavalent Soluble NY-ESO TCRs

Functional expression of the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR is performed in suspension-adapted HEK293 EBNA cells. HEK293-EBNA cells are cultured in serum-free Dulbecco's modified Eagle's medium (DMEM, high glucose (4.5 g/L) with 2 mM L-glutamine) at 37° C., 5% CO ₂ and 95% humidity.

For each transfection, HEK293-EBNA cells (3×10 ⁷ cells) are freshly seeded into 250 mL Erlenmeyer shaker flasks (Corning) from approximately 60% confluent cells. Transfections are performed using the FreeStyle MAX Cationic Lipid Base Reagent (Life Technologies) according to the supplier's guidelines. For expression of the ts-NY-ESO-1 TCR, 37.5 μg of total plasmid DNA (pTT5-ts-NY-ESO-1-TCRa and pTT5-ts-ESO-1-TCRβ vectors were each loaded with 18 μg). Either 75 μg of plasmid DNA is used, or different amounts of the two expression plasmids) are used per transfection. Similarly, for expression of os-NY-ESO-1 TCR, pTT5-os-NY-ESO-1-TCRa and pTT5-os-ESO-1-TCRβ were each transfected with 18.75 μg of plasmid DNA. use for Cells were harvested in fresh medium after transfection and cultured at 37° C., 5% CO 2 in an orbital shaker at 110 rpm for 4-8 days. Smaller scale transfections are performed in 6-well or 24-well plates as well.

Analysis of Expressed Soluble eTCR ² -BiTEs Secreted ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR protein molecules in the supernatant were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). ) directly or after protein purification. Protein samples and standards are prepared under both reducing and non-reducing conditions. SDS-PAGE was performed using cast minigels for protein electrophoresis on the Mini-PROTEAN Tetra cell electrophoresis system (Bio-Rad). Coomassie blue dye was used to stain proteins in SDS-PAGE gels.

Purification of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR Protein Molecules Purification of soluble ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from cell supernatants in two steps . The first step uses immobilized metal affinity chromatography (IMAC) with nickel-loaded nitrilotriacetic acid (NTA) agarose resin (HisPur Ni-NTA Superflow agarose-Thermo fisher). Binding and washing buffers consist of Tris-buffered saline (TBS), pH 7.2, containing low concentrations of imidazole (10-25 mM). Elution and recovery of His-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from the IMAC column is achieved by washing with high concentrations of imidazole (>200 mM). Eluted protein fractions are analyzed by SDS-PAGE and fractions containing the protein of interest are pooled. Pooled protein fractions are used directly in binding assays or further purified in a second step involving size exclusion chromatography (SEC). Superdex 200 increase pre-packed columns (Gelifesciences) are used to separate out monomeric, oligomeric and any aggregated forms of the target protein.

Surface Plasmon Resonance Specific binding and affinity analyzes of the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR to their pMHC are performed using BIAcore. Briefly, purified histidine-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR proteins are captured on the sensor surface via Ni ²⁺ chelation of nitrilotriacetic acid (NTA). Different concentrations of analyte solutions containing NY-ESO pep(SLLMwiTQv)-MHC (ProImmune) were injected and binding signals were monitored.

Example 2 Generation of Tetravalent and Octavalent Soluble NY-ESO TCR-IL2 Fusion Molecules To Express ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 Protein Complexes DNA encoding the required domains is synthesized as described above and cloned into the expression vector pTT5. Diagrammatic depictions of the domains within the TCR αβ chain for ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 are shown in FIGS. 7 and 8, and the amino acid sequences are shown in FIGS. It is shown in FIG.

Expression, purification and characterization of ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 are performed as described above.

Example 3: High Yield of Tetramer Secretion Another HEK293-T cell line encoding 17 (FIGS. 14 and 15) was generated.

Protein expression occurred and the protein was secreted from the cell line. Samples of the medium in which the cells were incubated were subjected to PAGE under denaturing conditions (SDS-PAGE) or PAGE under native conditions (no SDS). The former was further subjected to reducing conditions (using mercaptoethanol), but the latter was not.

The reduced gel showed a distinct band at the expected monomer size (Figure 16). Surprisingly, the non-reducing, native gel showed no detectable bands at the monomer, dimer or trimer sizes, instead showing strong bands at the tetramer size and tetramer size. SEC indicated that a very high yield of the mer was obtained, which was confirmed to be of high purity.

For Quad 16, the tetramer peak from SEC was run on SDS-PAGE and the resulting band was excised for mass spectrometry analysis. Data were obtained by trypsin digestion and p53 was detected at 100% protein. This is conclusive evidence that secreted Quad 16 was multimerized.

Example 4: Intracellular protein expression of the extracellular portion of TCR fused to NHR2 TP Expression vector All DNA fragments were synthesized into the expression vector pEF/myc/cyto (Invitrogen) by Twist Bioscience (California). cloned. The schematics and sequences of the synthesized DNA fragments and Quad polypeptides are shown in Figure 21 and in the Sequence Listing herein.

Preparation of DNA Lyophilized plasmid DNA synthesized by Twist Bioscience was resuspended in MQ water to a concentration of 50 ng/μl. 50 ng of DNA was transformed into 50 μl of competent DH5a cells using the conventional heat shock method. Cells were plated on LB agar plates containing 100 μg/mL ampicillin and grown overnight at 37°C. Individual colonies were picked and grown overnight at 220 rpm and 37°C. DNA was purified from the cells using the QIAprep Spin Miniprep Kit according to the manufacturer's instructions (Qiagen).

Transfection into HEK293T cells Briefly, HEK293T cells were maintained in high glucose DMEM supplemented with 10% FBS and Pen/Strep. Cells were seeded at 6×10 ⁵ cells per well of 6-well plates in 2 ml medium and allowed to adhere overnight at 37° C., 5% CO2. 7.5 μl Lipofectamine 2000 was diluted in 150 μl OptiMem and incubated for 5 minutes at room temperature. Plasmid DNA (2.5 μg) was diluted in 150 μl of OptiMem. The diluted DNA was combined with diluted Lipofectamine 2000, mixed gently and incubated at room temperature for 20 minutes. 300 μl of complex was added to one well of a 6-well plate. If the analysis required the medium to be serum-free, the medium was aspirated and replaced with CD293 medium 6 hours after transfection. Cells were incubated at 37°C, 5% CO2 for 48 hours before analysis.

Therefore, different formats of TCR-linked NHR2 tetramerization domain (TD) constructs (Quad) were transfected into HEK293T cells. Quad 3 and Quad 4 (structures represented schematically in FIGS. 1 and 3), which resemble the tetravalent format of the TCR, and Quad 12 and Quad 13, which resemble the octavalent format of the TCR (schematically shown in FIGS. 2 and 4). Schematic representation) were transfected for protein expression analysis. To confirm intracellularly expressed protein, protein samples were prepared from transfected HEK293T cells as follows. Briefly, cells were washed once with 2 ml PBS and then aspirated. 150 μl of trypsin-EDTA (0.05%) was added to each well and the cells were incubated at room temperature for approximately 1 minute. The plate was tapped to dislodge any strongly adherent cells. 850 μl of medium was added to each well to inactivate trypsin. Cells were transferred to a 1.5 ml Eppendorf and centrifuged at 1,000 rpm for 5 minutes. The supernatant was aspirated and the pellet was stored on ice. Cells were resuspended and diluted 1/200 in 400 μl cell lysis buffer (10 mM Tris, pH 7.5, 1% SDS) containing Protease Inhibitor Cocktail Set III (Calbiochem). Samples were vortexed vigorously and incubated on ice for 20 minutes. Cells were sonicated using a Branson Ultrasonics Sonifier™ (Thermo Fisher Scientific). The amplitude was set to 30% and the cells were sonicated for a total of 24 seconds (4 times 6 seconds on, 3 seconds off). Total protein concentration was quantified using the Pierce BCA Protein Assay Kit™ according to the manufacturer's instructions. 100 μg was diluted with MQ water to a volume of 80 μl. 20 μl of 5× SDS loading buffer was then added to give a 1 mg/ml sample. Samples were incubated at 95°C for 5 minutes prior to SDS-PAGE and Western blot analysis.

Protein samples were separated on SDS-PAGE under denaturing conditions. Typically, 25 μg of total cell lysate (25 μl) was run on the gel for Western blot analysis. 5 μl of PageRule Prestained 10-180 kDa Protein Ladder was run on the gel next to the protein samples. Gel run was performed in Tris-glycine buffer containing 0.1% SDS. A constant voltage of 150 volts was used and the gel was run for approximately 70 minutes until the dye front had fully migrated.

SDS-PAGE (15% Bis-Tris) gels were prepared using the following resolving and stacking gels.

Separation gel:
・5 ml 30% bis-acrylamide ・2.6 ml 1.5 M Tris (pH 8.8)
・50 μl 20% SDS
・100 μl 10% APS
・10 μl TEMED
・2.2ml MQ water

Concentrated gel:
・0.75 ml 30% bis-acrylamide ・1.25 ml 1.5 M Tris (pH 8.8)
・25 μl 20% SDS
・50 μl 10% APS
・5 μl TEMED
・2.9ml MQ water

Western blotting was performed for specific and sensitive detection of protein expression of TCR-NHR2 TD fusion proteins from Quad 3, Quad 4, Quad 12 and Quad 13. Proteins separated by SDS-PAGE were transferred to Amersham Hybond™ 0.45 μM PVDF membranes as follows. Briefly, Amersham Hybond 0.45 μM PVDF membranes were activated with MeOH for approximately 1 minute and rinsed with transfer buffer (25 mM Tris, 190 mM glycine, 20% MeOH) before use. A stack of sponge, filter paper, gel, membrane, filter paper, sponge was prepared and placed in a cassette for transfer. Transfers were performed at 280 mA for 75 minutes on ice. Membranes were incubated in blocking buffer (TBST, 5% milk powder) for approximately 2 hours. Membranes were washed briefly with TBST and then incubated overnight at 4° C. with anti-human IgG HRP (Thermo, 31410) diluted 1/2500 in TBST, 1% milk powder. After extensive washing of the membrane (3 washes with TBST, 15 min each), the color was developed using Pierce ECL Western Blotting Substrate.

Using an anti-human IgG detection antibody to probe Western blots, specific protein bands of predicted molecular weight were identified as Quad 3 (46.1 kDa), Quad 4 (46.4 kDa), Quad 12 (47.8 kDa). ) and Quad 13 (48.1 kDa) (FIGS. 17A and 17B). These data confirm intracellular protein expression of the TCR-NHR2 TD fusion protein in HEK293T cells.

A distinct single band could be detected for all Quads analyzed, indicating that the fusion of TRVβ-TRVβ-IgG1-CH1 (+/- IgG hinge domain) with NHR2 TD is stable. These expression data also confirm that the tetravalent (Quad 3 and Quad 4) and octavalent (Quad 12 and Quad 13) molecules exemplified in this example can be assembled.

The difference between Quad 3 and Quad 4 is the presence of a small peptide linker (G4S) located between the IgG1 CH1 domain and the NHR2 TD. This is also true for Quad 12 and Quad 13, Q13 containing a peptide linker between the IgG1 CH1 domain and the NHR2 TD. Expression data show that peptide linkers do not result in protein expression. However, it may be desirable to include a peptide linker in these TCR-NHR2 TD formats to aid in antigen binding and/or stabilization of the multimerization complex.

Example 5: Soluble protein expression of extracellular portion of TCR fused to NHR2 TP In Example 4, it was shown that the TCR-NHR2 TD fusion protein was expressed intracellularly in HEK293T cells. Here again, Quad 3, Quad 4, Quad 12 and Quad 13 were used to demonstrate soluble expression of these fusion proteins. Quad 3, Quad 4, Quad 12 and Quad 13 were transfected into HEK293T cells and soluble proteins from cell supernatants were enriched as described above. Briefly, 48 hours post-transfection, media was harvested and centrifuged at 2,000 rpm for 5 minutes to remove any cells or debris. Typically, 500 μl of medium was concentrated to 100 μl using an Amicon™ Ultra 0.5 Centrifugal Unit with a MWCO of 10 kDa. 25 μl of 5×SDS loading buffer was added to the samples and then incubated at 95° C. for 5 minutes prior to gel/western blot analysis. Concentrated protein samples were separated on SDS-PAGE gels and transferred to Amersham Hybond 0.45 μM PVDF membranes. Western blotting and protein detection were performed using anti-human IgG HRP using the methods described above.

Protein samples enriched and prepared from cell supernatants were analyzed on Western blots corresponding to Quad 3 (46.1 kDa), Quad 4 (46.4 kDa), Quad 12 (47.8 kDa) and Quad 13 (48.1 kDa). (Figs. 18A and 18B). Western blot expression data clearly demonstrate soluble expression of the TCR-NHR2 TD fusion protein in HEK293T. These data are the first reports demonstrating soluble expression of TCR-NHR2 TD fusion proteins expressed in eukaryotic cells such as HEK293T cells.

Detection of soluble protein expression from both tetrameric (Quad 3 and Quad 4) and octameric (Quad 12 and Quad 13) TCR-NHR2 TD formats has demonstrated the potential application of NHR2 TDs in a wide variety of contexts. Emphasize. The use of NHR2 TD fusion molecules can be used for the preparation of therapeutic molecules and protein molecules for use in diagnostics and imaging.

Example 6: Intracellular Protein Expression of Antibody Fragments Fused to NHR2 TD To further illustrate the versatility of NHR2 TD, several different antibody fragment formats fused to NHR2 TD were constructed to test expression in HEK293T cells. bottom.

Quad 14 and Quad 15 contain antibody VH domains fused to NHR2 TD with or without a peptide linker located between VH and NHR2 TD schematically depicted in FIGS. The VH domains in Quad 14 and Quad 15 are specific for GFP (green fluorescent protein). Several other versions of this format were also constructed and tested with VHs specific for therapeutically useful drug targets. The binding domain sequences are listed in Table 4. Some of these include Quad 34 (TNFa specific), Quad 38 (VEGF specific), Quad 40 (EGFR specific) and Quad 44 (CD38 specific).

Quad 38 and Quad 44 were further developed containing additional binding arms with inclusion of a second VH domain specific for EGFR and CD138 resulting in Quad 42 and Quad 46, respectively. Quad 42 and Quad 46 represent bispecific molecules with the ability to multimerize through the NHR2 TD domains forming bispecific tetramers.

In another example, an effector molecule (human IL2) is linked to the C-terminus of Quad 14 and Quad 15 resulting in Quad 18 and Quad 19, whereby the VH-NHR2-IL2 molecule is tetravalent and bifunctional. becomes.

In another example, an antibody Fab fragment (VH-CH1) was linked to NHR2 TD (Quad 23 and Quad 24) as schematically depicted in FIG. Quad 23 and Quad 24 represent tetravalent Fab molecules when co-expressed or mixed and assembled in vitro with a second chain containing an immunoglobulin light chain (eg Quad 25).

In yet another example, the human IgG1 hinge domain is included in Quad 23 and Quad 24, designated Quad 26 and Quad 27, and depicted schematically in FIG. Quad 26 and Quad 27 represent octavalent Fab molecules when co-expressed or mixed and assembled in vitro with a second chain containing an immunoglobulin light chain domain (eg Quad 25).

The following Quad vectors, all His-tagged, Quad 14, Quad 15, Quad 18, Quad 19, Quad 23, Quad 24, Quad 26, Quad 27, Quad 34, Quad 38, Quad 40, Quad 42, Quad 44 and Quad 46 was transfected into HEK293T cells. Protein samples were prepared from whole cell extracts as described above, separated on SDS-PAGE and transferred to Amersham Hybond 0.45 μM PVDF membranes. Specific protein expression was detected using anti-His HRP (Sigma, A7058) diluted 1/2500 in TBST, 1% milk powder as a probe.

All the different Specific protein expression could be detected in whole cell extracts for the antibody-NHR2 TD fusion protein (FIGS. 19A-D). Interestingly, Quad 18 and Quad 19, which contain an effector domain (IL2) fused to the C-terminus of the NHR2 TD domain in addition to the VH-binding domain fused to the N-terminus of the NHR2 TD, showed good protein expression. .

Expression of Quad 23 and Quad 24 polypeptides can use NHR2 TD to form tetravalent antibody Fab molecules when co-expressed or mixed in vitro with a partner soluble Quad molecule (e.g., Quad 25). to emphasize Similarly, expression of Quad 26 and Quad 27 containing the human IgG1 hinge domain was co-expressed or mixed in vitro with a partnered soluble second partner chain (e.g., Quad 25) using NHR2 TD. It is emphasized that at times an octavalent antibody Fab molecule can be formed.

Quad 42 and Quad 46 bispecific molecules containing additional VH domains fused to the C-terminus of the NHR2 TD domain also showed good protein expression. These data highlight the versatility of the NHR2 TD domain and its ability to be fused to different binding and effector molecules to develop a wide range of protein formats. The data also suggest that protein molecules can be fused to both the N-terminus and C-terminus of NHR2 TD, allowing the development of bispecific, multivalent protein molecules.

Example 7 Multivalent Assembly of Antibody Fragments Fused to NHR2 TD NHR2 TD is involved in the oligomerization of ETO into tetrameric complexes. As shown in Examples 4-6, NHR2 TD domains can be used to fuse binding domains and effector molecules at the N-terminus or C-terminus or both N-terminus and C-terminus without affecting expression. Binding domains can be TCR variable and constant domains, antibodies and antibody fragments or effector molecules such as IL2. NHR2 TD is also able to express protein in soluble format when fused to NHR2 TD, even though it is part of an intracellular protein that is naturally only expressed intracellularly (Fig. 18).

To demonstrate whether NHR2 TDs retain the ability to oligomerize when fused to the binding domain, Quad 14 and Quad 15 were expressed in HEK293T cells and protein samples were isolated from whole cell extracts as described above. prepared. Protein samples were separated on PAGE gels under denaturing and non-denaturing (native) conditions. Native gels were prepared using the protocol above without SDS. Proteins from PAGE gels were transferred to Amersham Hybond 0.45 μM PVDF membranes. An anti-human IgG HRP detection antibody was used as a probe to detect specific protein expression.

As expected, under denaturing conditions, the expression of VH-NHR2 TDs from Quad 14 and Quad 15 can be seen as monomeric, with specific protein bands at the predicted molecular weights (22 and 22.3 kDa). can be detected (FIG. 20A). Under non-denaturing and thus native conditions, interestingly, no monomers or dimers of VH-NHR2 TDs from Quad 14 and Quad 15 can be detected (Fig. 20B). Only high molecular weight protein bands that appear to be tetramers of VH-NHR2 TDs from Quad 14 and Quad 15 can be detected. Tetramer assembly appears to be highly efficient and pure, as judged by protein intensity and the absence of any detectable monomers and dimers of Quad 14 and Quad 15.

Combined with the data from Examples 4-7, there is conclusive evidence that NHR2 TD is highly versatile and allows the fusion of various protein binding domains and effector molecules. NHR2 TDs allow soluble expression of proteins from eukaryotic cells such as HEK293T cells and form highly stable and pure tetrameric molecules.

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Claims (50)

A protein multimer of at least first, second, third and fourth copies of an effector domain (e.g., protein domain) or peptide, wherein said multimers associate together each engineered protein multimerizing by a third and fourth self-associating tetramerization domain (TD), each tetramerization domain comprising one or more copies of said protein domain or peptide; A multimer composed of multiple polypeptides. 2. The multimer of claim 1, which is a tetramer or octamer of said domain or peptide. 3. The multimer of any of claims 1 or 2, comprising a tetramer or octamer of an immunoglobulin superfamily binding site. 4. The multimer of claim 3, wherein said binding site comprises a first variable domain paired with a second variable domain. Each polypeptide comprises first and second copies of said protein domain or peptide, said polypeptide comprising (in N-terminal to C-terminal direction): (i) said first copy - TD - said second (ii) TD- and said first and second copies; or (iii) said first and second copies-TD. . Claims 1- wherein said TD is an NHR2 TD and said domain or peptide is not an NHR2 domain or peptide; or said TD is a p53 TD and said domain or peptide is not a p53 domain or peptide 6. The multimer according to any one of 5. said engineered polypeptide comprises one or more copies of a second type of protein domain or peptide, wherein said second type of protein domain or peptide is different from said first protein domain or peptide , the multimer according to any one of claims 1 to 6. A multimer according to any one of claims 1 to 7, wherein said domain is an immunoglobulin superfamily domain. 9. The multimer of any one of claims 1-8, wherein said main domain or peptide is an antibody variable or constant domain, a TCR variable or constant domain, an incretin, an insulin peptide, or a hormone peptide. said multimer comprises first, second, third and fourth identical copies of said engineered polypeptide, said polypeptide comprising a TD and one of said protein domains or peptides, provided that 10. A multimer according to any one of claims 1 to 9, comprising no more than 1), 2 (but no more than 2) or more copies. 11. The multimer of any one of claims 1-10, wherein said engineered polypeptide comprises an antibody or TCR variable domain (V1) and an NHR2 TD. (ii) V1-optional linker-NHR2 TD-optional linker-V2 or (iii) V1-optional linker-V2-optional linker-NHR2 TD, wherein V1 and V2 are TCR variable domains and are the same or different, or V1 and V2 are antibody 12. The multimer of claim 11, which is a variable domain and is the same or different. 13. The multimer of claim 12, wherein V1 and V2 are antibody single variable domains. Each engineered polypeptide comprises (from N-terminus to C-terminus) V1-optionally present linker-TD, where V1 is an antibody or TCR variable domain, and each engineered polypeptide comprises , with each second engineered polypeptide comprising V2, wherein V2 is an antibody or TCR variable domain that pairs with V1, respectively, to form an antigen or pMHC binding site; and 12. The multimer of claim 11, wherein one polypeptide comprises antibody Fc or comprises antibody CH1, and the other polypeptide comprises antibody CL paired with CH1. said TD comprises (i) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 10 or 126; or (ii) an amino acid sequence identical to or at least 80% identical to SEQ ID NO: 120 or 123 , the multimer according to any one of claims 1 to 14. Abciximab; Rituxan™; Rituximab; Zenapax™; Daclizumab; Alemtuzumab; Zevalin™; Ibritumomab; Humira™; Adalimumab; Xolair™; Omalizumab; Bexar™; Avastin™; Bevacizumab; Tysabri™; Natalizumab; Actemra™; Tocilizumab; Cimzia™; Certolizumab; Simponi™; Golimumab, Ilaris™; Canakinumab; Stelara™; belimumab; Yervoy™; ipilimumab; ADCETRIS™; brentuximab; Trastuzumab; comprising a tetramer or octamer of the antigen-binding site of an antibody selected from the group consisting of Keytruda™, Opdivo™, Gazyva™ and Obinutuzumab. 2. The multimer according to item 1. An isolated tetramer or octamer, or a plurality of said tetramers or octamers, of a TCR binding site, insulin peptide, incretin peptide or peptide hormone. (a) is soluble in an aqueous solution;
(b) secretable from a eukaryotic cell and/or (c) an expression product of a eukaryotic cell,
A multimer, tetramer or octamer according to any one of claims 1-17. (a) a tetramer or octamer of a TCR V domain or TCR binding site that is soluble in aqueous solution;
(b) a tetramer or octamer of an antibody single variable domain that is soluble in aqueous solution;
(c) a tetramer or octamer of a TCR V domain or TCR binding site that can be expressed intracellularly and/or extracellularly by HEK293 cells, or (d) intracellularly and/or extracellularly by HEK293 cells Tetramers or octamers of antibody variable domains that are expressible. A multimer, tetramer or octamer according to any one of claims 1 to 19, wherein said tetramer or octamer is bispecific for antigen or pMHC binding. . A multimer, tetramer or octamer according to any one of claims 1 to 20, wherein said domains are identical. 22. A multimer, tetramer or octamer according to any one of claims 1 to 21 comprising eukaryotic glycosylation. 23. The multimer, tetramer or octamer of claim 22, wherein said cells are HEK293 cells. A plurality of multimers, tetramers or octamers according to any one of claims 1-23. A pharmaceutical composition comprising a multimer, tetramer or octamer according to any one of claims 1-24 and a pharmaceutically acceptable carrier, diluent or excipient. Cosmetics, foodstuffs, beverages, cleaning products, detergents comprising a multimer, tetramer or octamer according to any one of claims 1-24. Said engineered (and optionally isolated) polypeptide or (optionally isolated) unit of a multimer, tetramer or octamer according to any one of claims 1-26 Quantity. (N-terminal to C-terminal direction):
(a) TCR V1-TCR C1-antibody CH1-optionally present linker-TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Cβ;
(iii) V1 is Vγ and C1 is Cγ, or (iv) V1 is Vδ and C1 is Cδ,
or (b) TCR V1-antibody CH1-optionally present linker-TD,
(i) V1 is Vα;
(ii) V1 is Vβ;
(iii) V1 is Vγ, or (iv) V1 is Vδ,
or (c) antibody V1-antibody CH1-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL,
or (d) comprising antibody V1-optionally present antibody CH1-antibody Fc-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL,
or (e) antibody V1-antibody CL-optionally present linker-TD,
(i) V1 is VH, or (ii) V1 is VL,
or (f) TCR V1-TCR C1-optionally present linker-TD,
(i) V1 is Vα and C1 is Cα;
(ii) V1 is Vβ and C1 is Cβ;
(iii) V1 is Vγ and C1 is Cγ, or (iv) V1 is Vδ and C1 is Cδ,
An engineered (and optionally isolated) polypeptide (P1). Said engineered polypeptide P1 is paired with a further polypeptide (P2), P2 (in the N-terminal to C-terminal direction):
(g) comprising TCR V2-TCR C2-antibody CL, wherein P1 is according to (a) as recited in claim 28, and (i) P1 is according to (a)(ii) , V2 is Vα and C2 is Cα,
(ii) if P1 is according to (a)(i), then V2 is Vβ and C2 is Cβ;
(iii) if P1 is due to (a)(iv), then V2 is Vγ and C2 is Cγ, or (iv) if P1 is due to (a)(iii), V2 is Vδ and C2 is Cδ,
or (h) TCR V2-antibody CL, where P1 is according to (b) as recited in claim 28, and (i) P1 is according to (b)(ii), V2 is Vα,
(ii) V2 is Vβ if P1 is according to (b)(i);
(iii) V2 is Vγ if P1 is due to (b)(iv), or (iv) V2 is Vδ if P1 is due to (b)(iii);
or (i) comprising antibody V2-CL, wherein P1 is according to (c) as recited in claim 28, and (i) P1 is according to (c)(ii), where V2 is or (ii) V2 is VL if P1 is due to (c)(i);
or (j) antibody V2 - optionally comprising CL, wherein P1 is according to (d) as described in claim 28, and (i) P1 is according to (d)(ii) or (ii) V2 is VL if P1 is due to (d)(i);
or (k) comprising antibody V2-CH1, wherein P1 is according to (e) as recited in claim 28, and (i) P1 is according to (e)(ii), where V2 is or (ii) V2 is VL if P1 is due to (e)(i);
or (l) includes TCR V2-TCR C2, P1 is according to (f) recited in claim 28, and (i) P1 is according to (f)(ii), then V2 is Vα and C2 is Cα,
(ii) V2 is Vβ and C2 is Cβ if P1 is according to (f)(i);
(iii) if P1 is due to (f)(iii), then V2 is Vγ and C2 is Cγ, or (iv) if P1 is due to (f)(iv), V2 is Vδ and C2 is Cδ,
29. A polypeptide according to claim 28. A multimer of P1 paired with P1 as defined in claim 28 or P2 as defined in aspect 29, or a plurality of said multimers, optionally wherein said multimers are A multimer or multimers as described in any one of paragraphs. 30. A nucleic acid encoding the engineered polypeptide or monomer of any one of claims 27-29, optionally comprising an expression vector for expressing said polypeptide. nucleic acids. 31, for intracellular and/or secretory expression of a multimer, tetramer, octamer, engineered polypeptide or monomer according to any one of claims 1-24. A eukaryotic host cell containing a nucleic acid or vector of 32. Use of the nucleic acid or vector of claim 31 in a method for producing protein multimers for producing intracellularly expressed and/or secreted multimers, wherein the method comprises an authentic A use comprising expressing said multimer in a nuclear cell and/or secreting said multimer from said eukaryotic cell. Use of a nucleic acid or vector according to claim 31 in a method for producing protein multimers for producing glycosylated multimers in a eukaryotic cell comprising said nucleic acid or vector. (i) a eukaryotic cell line encoding an engineered polypeptide according to any one of claims 27-29; and (ii) a multimer as defined in any one of claims 1-24, A mixture containing tetramers or octamers. 36. The mixture of claim 35, wherein said cell line is in a medium comprising secreted products of said cells, said secreted products comprising said multimers, tetramers or octamers. A multimer, tetramer or octamer according to any one of claims 1 to 24 for medical use. (a) soluble and/or intracellular expression of a TCR V-NHR2 TD or TCR V-p53 TD fusion protein expressed in a eukaryotic cell, optionally comprising isolating a plurality of said multimers; , a method for producing TCR V domain multimers;
(b) soluble and/or intracellular expression of the antibody V-NHR2 TD or V-p53 TD fusion protein expressed in eukaryotic cells, optionally isolating a plurality of said multimers; a method of producing antibody V domain multimers,
(c) soluble and/or intracellular expression of an incretin peptide-NHR2 TD or incretin peptide-p53 TD fusion protein expressed in eukaryotic cells such as HEK293T cells, optionally comprising a plurality of said multimers; or (d) soluble and/or peptide hormone-NHR2 TD or peptide hormone-p53 TD fusion proteins expressed in eukaryotic cells such as HEK293T cells. or a method of producing a peptide hormone multimer comprising intracellular expression and optionally isolating a plurality of said multimers. Use of a self-associating tetramerization domain (TD) in a method for producing tetramers of polypeptides to produce tetramers in higher yields than monomeric and/or dimeric polypeptides. Engineered Polypeptides in Methods for Producing Tetramers of Polypeptides Comprising Multiple Copies of a Protein Domain or Peptide to Produce Tetramers in Higher Yields than Monomeric and/or Dimeric Polypeptides wherein said engineered polypeptide comprises one or more copies of said protein domain or peptide and further comprises a self-associating tetramerization domain (TD). Self-associating tetramerization domains (TD) in methods for producing tetramers of polypeptides to produce multiple tetramers that are not mixed with monomers, dimers or trimers use. Engineered methods for producing tetramers of a polypeptide comprising multiple copies of a protein domain or peptide to produce tetramers that are not mixed with monomers, dimers or trimers. wherein said engineered polypeptide comprises one or more copies of said protein domain or peptide and further comprises a self-associating tetramerization domain (TD). Use according to any one of claims 39 to 42, wherein said yield of tetramer is at least 10 times said yield of monomer and/or dimer. Use according to any one of claims 39 to 43, wherein the ratio of tetramer produced in said process:monomer and/or dimer produced is at least 90:10. Use according to any one of claims 39 to 44, wherein each monomer has a size of 40 kDa or less. Use according to any one of claims 39 to 45, wherein each tetramer has a size of 150 kDa or less. Use according to any one of claims 39 to 46, wherein said method comprises expressing said tetramer from a eukaryotic cell line. (a) a TCR extracellular domain;
A multivalent heterodimeric soluble T-cell receptor capable of binding to pMHC complexes, comprising (b) an immunoglobulin constant domain, and (c) the NHR2 multimerization domain of ETO. A multimeric immunoglobulin comprising (i) an immunoglobulin variable domain and (ii) an NHR2 multimerization domain of ETO. A method of assembling into soluble multimeric polypeptides, comprising:
(a) providing a monomer of said multimeric polypeptide fused to the NHR2 domain of ETO; and (b) associating multiple copies of said monomer, thereby forming a multimeric soluble polypeptide. A method that includes obtaining.

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